CN114410628A - Acid-resistant expression cassette, application thereof and method for high-throughput screening of stress-resistant expression cassette for microbial fermentation - Google Patents

Acid-resistant expression cassette, application thereof and method for high-throughput screening of stress-resistant expression cassette for microbial fermentation Download PDF

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CN114410628A
CN114410628A CN202111585143.XA CN202111585143A CN114410628A CN 114410628 A CN114410628 A CN 114410628A CN 202111585143 A CN202111585143 A CN 202111585143A CN 114410628 A CN114410628 A CN 114410628A
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acid
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expression cassette
promoter
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林章凛
杨晓锋
姚栩荣
刘鹏
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South China University of Technology SCUT
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Abstract

The invention discloses an acid-resistant expression cassette, application thereof and a method for high-throughput screening of a stress-resistant expression cassette for microbial fermentation. The acid-resistant expression cassette consists of more than one promoter, more than one stress-resistant gene and more than one terminator, wherein the stress-resistant gene is selected from a gadE gene, a sodB gene, a katE gene and a hdeB gene, and an acid-response asr promoter library is constructed. The invention also provides a method for high-throughput screening of stress-resistant expression cassettes for microbial fermentation. The method relates the growth rate and the fermentation performance, avoids neglecting the influence of the balance relation between the growth and the fermentation performance through growth evaluation, adopts high-flux micro fermentation to evaluate the stress resistance and the fermentation performance after two-step screening, and finally screens a small amount of stress resistance expression cassettes with the best utilization prospect for subsequent evaluation in an industrial fermentation tank, thereby avoiding directly evaluating all the acid resistance expression cassettes through mass fermentation and improving the screening effect and efficiency.

Description

Acid-resistant expression cassette, application thereof and method for high-throughput screening of stress-resistant expression cassette for microbial fermentation
Technical Field
The invention relates to the field of genetic engineering and synthetic biology, in particular to an acid-resistant expression cassette, application thereof and a method for high-throughput screening of a stress-resistant expression cassette for microbial fermentation.
Background
The organic acid fermentation industry in China is an important part of the biological industry, the development is rapid in recent years, and however, various problems to be solved in the aspects of production economy, environmental protection and the like exist.
In the fermentation process, industrial microorganisms can gradually accumulate a large amount of acidic products or byproducts, the fermentation environment is continuously acidified, the growth and metabolism of the industrial microorganisms are seriously inhibited in the acidic environment, and the production capacity is reduced. Therefore, in the actual industrial fermentation process (such as amino acid fermentation), a large amount of alkali (such as ammonia water) is used for neutralizing acidic substances generated by fermentation, and neutral fermentation is maintained. In order to obtain the product of the acidic substance, a large amount of acid is needed after neutral fermentation, and a large amount of acid and alkali can generate a large amount of industrial wastewater. If the acid resistance of industrial microorganisms can be improved, the acid-resistant industrial microorganisms are utilized for fermentation under the acidic condition, so that the acid and alkali usage amount in the fermentation process can be effectively reduced, the wastewater discharge is reduced, meanwhile, the mixed bacteria pollution is inhibited, the related energy consumption is reduced, huge economic benefits are generated, and the energy conservation and emission reduction are promoted.
The research on the stress resistance of microorganisms at home and abroad, such as acid resistance, high temperature resistance, high osmotic pressure resistance and the like, mainly comprises traditional mutagenesis, domestication, simple gene cloning and knockout and the like, but the industrialization efficiency of the methods is lower. In recent years, new methods based on regulatory genes are developed at home and abroad for improving the stress resistance of microorganisms, such as global transcription machinery gTME, artificial transcription factors, global regulatory factor Engineering and the like, and the technical methods and ideas of microorganism stress resistance modification are expanded (Lin, Z., Y.Zhang, and J.Wang, Engineering of genetic regulation and Engineering strain tolerance. Biotechnical Adv,2013.31(6): p.986-91.).
In recent years, synthetic biology has made significant advances in conceptual theory, functional applications, and process technology. On the basis of known stress resistance related structural genes and regulatory genes, the stress resistance related structural genes and the regulatory genes can be artificially modified and integrated by using a synthetic biology method, so that a standardized stress resistance meta-expression cassette is designed, and the microorganism is endowed with high-efficiency stress resistance and is used for the actual production of industrial microorganisms. In Regulating Gene Expression, a promoter is a crucial element, and an Expression cassette is regulated by using an environmentally-responsive promoter, so that the Expression level of a Gene can reach a proper amount of Expression at a proper time, excessive energy and substances are prevented from being used for synthesis of other genes, and the stress tolerance is effectively improved, thereby improving the yield of industrial strains (Kent, r., and Dixon, n.contextual Tools for Regulating Gene Expression in bacteria.trends Biotechnol,2020.38,316-333) (Rottinghaus, a.g., amofel, m.b., and Moon, t.s.sensing in rt Engineered biology.biotechnol J,2020.15, e 1900319).
Coli has three major types of Acid Resistance (AR) mechanisms (Capitani, G., et al., Crystal structure and functional analysis of Escherichia coli substrate EMBO J,2003.22(16): p.4027-37.). Among them, the second type of antacid system (AR2) is a very important and well-defined system. The second type of acid-resistant system (AR2) consists of glutaminase Ybas (Hersh, B.M., et al, A glutamate-dependent acid resistancegene in Escherichia coli.J.Bacteriol, 1996.178(13): p.3978-81.), glutamate decarboxylases GadA and GadB (Ma, Z., Gong, S., Richard, H., Tucker, D.L., Conway, T., and Foster, J.W.GadE (Yohe) activators carboxlase-dependent acid resistance in Escherichia coli K-12.Mol Microbiol,2003.49: 1309-. Ybas of this system converts glutamine to glutamate, releasing NH3Neutralizing intracellular protons; the GAdA and the GadB enable glutamic acid to generate decarboxylation reaction to generate gamma-aminobutyric acid and consume intracellular protons; GadC intakes glutamine and glutamic acid, excreting glutamic acid and aminobutyric acid. GadE can up-regulate the expression of AR 2-related genes, a key regulator for increasing the acidity of strains (Dahl, j.u., Koldewey, p., Salmon, l., Horowitz, s., Bardwell, j.c., and Jakob, u.hde functions as an acid-protective chain in bacteria, j Biol Chem,2015.290: 65-75.).
SodB and KatE are superoxide dismutase and catalase in the antioxidant system of Escherichia coli, and can eliminate ROS in Escherichia coli cells, reduce oxidative stress caused by acid stress, and reduce damage to important enzyme systems and DNA or RNA in cytoplasm (Lushchak, V.I.oxidative stress and mechanisms of protection against infection in bacteria. biochemistry (Mosc),2001.66:476-2O2-induced oxidative damage.Biochem Biophys Res Commun,2003.301:915-922.)。
HdeB is a periplasmic space molecular chaperone capable of protecting periplasmic space enzymes or proteins in moderate acid environments and preventing the destruction of key enzyme systems, thereby improving the acid stress survival rate of E.coli (Lushchak, V.I. oxidative stress and mechanisms of protection acquisition in bacteria. biochemistry (Mosc),2001.66: 476-489.).
Although the mechanism of the acid-resistant system gene of the Escherichia coli is studied in detail at present, the research of developing a standardized synthetic biological acid-resistant expression cassette by using the gene and an artificial promoter is not available at present, and a method for evaluating the acid-resistant performance of the acid-resistant expression cassette and the influence on the fermentation production performance step by step is not available.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides an acid-resistant expression cassette, a screening method and application thereof, which can be used for improving the acid-resistant and fermentation production performance of industrial microorganisms under industrial conditions.
The invention provides an antacid expression cassette and a screening method and application thereof, and particularly relates to the following six aspects.
In a first aspect, the invention provides an acid-responsive promoter mutation library, which consists of mutations in one promoter, and the reporter gene is selected from the mCherry gene. The promoter mutants of the present invention are subsequently invented as elements for the construction of expression cassettes. The promoter in the acid-response promoter bank is used as a regulation element in the acid-resistant expression cassette and can change the expression quantity of the acid-resistant regulation gene in response to the pH value of the environment.
The invention provides a screening method of an acid response promoter. The asr promoter in the acid-responsive promoter mutation library is shown as SEQ ID NO. 11-59, and the wild type of the acid-responsive asr promoter is shown as SEQ ID NO. 10.
In a second aspect, the present invention provides an expression cassette consisting of one or more promoters, one or more stress-resistant genes selected from the group consisting of gadE gene, sodB gene, katE gene and hdeB gene, and one or more terminators. The expression cassette can express the acid-resistant regulatory gene in the host cell, and can improve the acid resistance of the host cell after being introduced into the host cell, thereby improving the acid resistance of the host cell, such as the growth rate of an acid pressure environment and the fermentation performance under the acid pressure environment.
In the expression cassette of the present invention, a promoter, an acid-fast regulatory gene and a terminator are operably linked to achieve expression of the desired acid-fast regulatory gene in a host cell.
The gadE gene in the expression cassette of the invention encodes the amino acid sequence shown in SEQ ID NO 1. The sodB gene in the expression cassette of the invention encodes an amino acid sequence shown in SEQ ID NO. 2. The katE gene in the expression cassette of the invention encodes the amino acid sequence shown in SEQ ID NO 3. The hdeB gene in the expression cassette of the invention encodes an amino acid sequence shown in SEQ ID NO. 4.
The promoter in the expression cassette is selected from the promoters shown in SEQ ID NO. 11-14, asr promoter mutant P24E8, asr promoter mutant P10G6, asr promoter mutant P77C4 and asr promoter mutant P50G 1.
The terminator in the expression cassette of the invention is the rrnB terminator. Wherein the nucleotide sequence of the rrnB terminator is shown in SEQ ID NO 9.
The expression cassette of the present invention consists of, from 5 'to 3', a promoter selected from the group consisting of SEQ ID NO:11-13, a gene encoding gadE as shown in SEQ ID NO:5, and a rrnB terminator as shown in SEQ ID NO: 9. The nucleotide sequence of the expression cassette is shown in any one of SEQ ID NO 60-62.
The expression cassette of the present invention consists of, from 5 'to 3', a promoter selected from the group consisting of SEQ ID NO:11-13, a sodB gene encoding SEQ ID NO:6, and an rrnB terminator as shown in SEQ ID NO: 9. The nucleotide sequence of the expression cassette is shown in any one of SEQ ID NO 63-65.
The expression cassette of the present invention consists of, from 5 'to 3', a promoter selected from the group consisting of SEQ ID NO:11-13, a katE gene encoding SEQ ID NO:7, and rrnB terminator as shown in SEQ ID NO: 9. The nucleotide sequence of the expression cassette is shown in any one of SEQ ID NO 66-68.
The expression cassette of the present invention consists of, from 5 'to 3', a promoter selected from the group consisting of SEQ ID NO:11-13, a hdeB gene encoding the gene of SEQ ID NO:8, and an rrnB terminator of SEQ ID NO: 9. The nucleotide sequence of the expression cassette is shown in any one of SEQ ID NO 69-71.
The expression cassette of the present invention consists of, from 5 'to 3', a promoter selected from the group consisting of SEQ ID NO:11-13, the sodB and katE genes encoded by SEQ ID NO:6 and 7, and the rrnB terminator indicated by SEQ ID NO: 9. The nucleotide sequence of the expression cassette is shown in any one of SEQ ID NO 72-74.
The expression cassette of the present invention is composed of three parts, the first part from 5 'to 3' comprises asr promoter mutant selected from SEQ ID NO. 11-14, sodB gene encoding SEQ ID NO. 6 and rrnB terminator shown in SEQ ID NO. 9, the second part from 5 'to 3' comprises asr promoter mutant selected from SEQ ID NO. 11-14, katE gene encoding SEQ ID NO. 7 and rrnB terminator shown in SEQ ID NO. 9, and the third part from 5 'to 3' comprises asr promoter mutant selected from SEQ ID NO. 11-14, hdeB gene encoding SEQ ID NO. 8 and rrnB terminator shown in SEQ ID NO. 9. In some specific embodiments, the nucleotide sequence of the expression cassette is as set forth in any one of SEQ ID NOs 75, 76.
The expression cassette of the invention consists of four parts, the first part comprising, from 5 'to 3': 11-14, an asr promoter mutant encoding the amino acid sequence of SEQ ID NO:5 and the gadE gene shown in SEQ ID NO:9, and a second portion from 5 'to 3' comprising a sequence selected from the group consisting of SEQ ID NOs: 11-14, an asr promoter mutant encoding the amino acid sequence of SEQ ID NO:6 and the gene of sodB shown in SEQ ID NO:9, and a second portion comprising, from 5 'to 3', an rrnB terminator selected from the group consisting of SEQ ID NOs: 11-14, an asr promoter mutant encoding the amino acid sequence of SEQ ID NO:7 and the katE gene shown in SEQ ID NO:9, and a third portion comprising, from 5 'to 3', a rrnB terminator selected from the group consisting of SEQ ID NOs: 11-14, an asr promoter mutant encoding the amino acid sequence of SEQ ID NO:8 and the hdeB gene shown in SEQ ID NO:9 rrnB terminator. In some specific embodiments, the nucleotide sequence of the expression cassette is as set forth in any one of SEQ ID NOs 77, 78.
In the present invention, the "acid-resistant regulatory gene" refers to an acid-resistant protein encoding Escherichia coli, for example, a gene gadE gene encoding an acid-resistant system regulatory transcription factor gadE, genes sodB and katE encoding antioxidant proteins SodB and KatE, and a gene hdeB gene encoding a periplasmic space chaperone protein HdeB. Genes encoding other acid-resistant proteins of E.coli may also be used in the expression cassettes of the invention.
The acid-resistant regulatory gene in the expression cassette of the present invention is acid-resistant regulatory gene gadE of Escherichia coli. In one embodiment, the gadE gene encodes the wild-type transcription factor gadE shown in SEQ ID NO. 1.
The acid-resistant regulatory gene in the expression cassette of the invention is the wild-type antioxidant gene sodB of escherichia coli. In a specific embodiment, the sodB gene encodes the wild-type SodB represented by SEQ ID NO. 2.
In the present invention, the acid-resistant regulatory gene in the expression cassette of the present invention is the wild-type antioxidant gene katE of Escherichia coli. In one embodiment, the katE gene encodes the wild-type KatE shown in SEQ ID NO. 3.
The acid-resistant regulatory gene in the expression cassette of the invention is a wild-type molecular chaperone gene hdeB of Escherichia coli. In one embodiment, the hdeB gene encodes the wild-type hdeB as shown in SEQ ID NO. 4.
The promoter of the expression cassette comprises 4 asr promoter mutants P24E8, P10G6, P77C4 and P50G1 which have the sequences shown in SEQ ID NO. 11-14. The asr promoter mutants P24E8, P10G6, P77C4 and P50G1 are promoters having different promoter efficiencies in acid stress response.
The terminator sequence of the expression cassette of the invention is shown in the rrnB terminator of the 16S ribosomal RNA rrnB operon of SEQ ID NO 9. Various terminators which can terminate transcription of a target gene in a host cell are known in the art, and such terminators are also encompassed in the scope of the present invention.
The invention provides in another aspect an expression construct comprising an expression cassette of the invention. The expression constructs of the invention may be based on any suitable vector. Vectors for use in the expression constructs of the invention include those that replicate autonomously in the host cell, such as plasmid vectors; also included are vectors that are capable of integrating into and replicating with host cell DNA. Many suitable vectors for the present invention are commercially available. In a specific embodiment, the expression construct of the invention is constructed based on the commercial plasmid pACYC184(New England Biolab).
The invention also provides in a further aspect a recombinant host cell comprising an expression cassette of the invention or an expression construct of the invention. It is known in the art that genes from one organism can function in other organisms as well, by means of codon optimization and the like. Thus, the expression cassette of the present invention is not limited to use in E.coli.The recombinant host cells of the invention are preferably prokaryotic cells, more preferably bacterial cells, most preferably E.coli cells such as the MG1655 strain. Various methods for introducing expression cassettes or expression constructs into host cells are well known in the art, e.g., CaCl2Methods, electrotransformation, etc. Recombinant host cells comprising an expression cassette of the invention have improved acid resistance compared to corresponding control cells not comprising the expression cassette or expression construct. As used herein, "acid resistance" includes survival under acid shock and growth rate under acid stress conditions.
In a third aspect, the present invention provides an expression construct comprising an expression cassette of the invention.
In a fourth aspect, the invention provides a recombinant host cell comprising an expression cassette of the invention or an expression construct of the invention. The recombinant host cell is preferably a prokaryotic cell, more preferably a bacterial cell, most preferably an E.coli cell. The recombinant host cell of the invention has an increased acid resistance compared to a corresponding cell not comprising said expression cassette or expression construct. The acid resistance property is a growth rate under an acid stress condition.
In a fifth aspect, the present invention provides a method for producing an organic acid by microbial fermentation, the method comprising:
(a) introducing an expression cassette or expression construct of the invention into an organic acid-producing microorganism;
(b) fermenting the microorganism; and
(c) the resulting organic acid is harvested.
The organic acid-producing microorganism used in the method for producing an organic acid by microbial fermentation of the present invention is preferably a prokaryotic microorganism, more preferably a bacterium, and most preferably Escherichia coli. Organic acids that can be produced by the methods of the invention include amino acids (e.g., lysine, threonine, tryptophan, glutamic acid), succinic acid, citric acid, and lactic acid.
Preferably, the expression cassette is selected from the group consisting of SEQ ID NOS: 78-81. Preferably, the lysine-producing microorganism is lysine-producing Escherichia coli. More preferably, the lysine-producing microorganism is the E.coli SCEcL3 strain.
In a sixth aspect, the present invention also provides a method for high throughput screening of stress resistant expression cassettes for fermentation of microorganisms, said method comprising the steps of:
(a) introducing an expression cassette to be screened into the microorganism;
(b) evaluating the growth rate of the microorganism under stress pressure environment with a high throughput growth tester such as BioscreenC;
(c) evaluating the fermentation performance of the microorganism in a stress pressure environment by using a fermentation tank;
(d) identifying an expression cassette capable of increasing said growth rate and/or said fermentation performance of said microorganism as a stress resistant expression cassette that can be used for fermentation of a microorganism.
As used herein, "stress" refers to stress conditions that primarily affect the growth of a microorganism. The stress conditions for the microorganism can be determined by a person skilled in the art. For example, for E.coli, acid stress refers to a pH below 7.0 but above 4.0, e.g., pH5.0, pH 6.0.
Compared with the prior art, the invention has the following advantages:
the method for screening the anti-stress expression cassettes in high flux is a step-by-step comprehensive evaluation method, the growth rate and the fermentation performance are associated, the influence of neglecting the balance relation between the growth and the fermentation performance through growth evaluation is avoided, and the anti-stress expression cassettes with the best utilization prospect obtained through final screening are subjected to subsequent evaluation in an industrial fermentation tank, so that the direct large-scale fermentation evaluation of all acid-resistant expression cassettes is avoided, and the screening effect and efficiency are improved.
Drawings
FIG. 1 shows the construction of the plasmid pACYC184-Pasr library-mCherry-rrnBT.
FIG. 2 shows the construction of the Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT acid-resistant expression plasmid pACYC184- (P24E8/P77C4/P50G1) -gadE-rrnBT.
FIG. 3 shows the construction of the Pasr mutant (P24E8/P77C4/P50G1) -sodB-rrnBT acid-resistant expression cassette expression plasmid pACYC184- (P24E8/P77C4/P50G1) -sodB-rrnBT.
FIG. 4 shows the construction of the Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT antacid expression cassette expression plasmid pACYC184- (P24E8/P77C4/P50G1) -sodB-rrnBT.
FIG. 5 shows the construction of the Pasr mutant (P24E8/P77C4/P50G1) -hdeB-rrnBT antacid expression plasmid pACYC184- (P24E8/P77C4/P50G1) -hdeB-rrnBT.
FIG. 6 shows the construction of the Pasr mutant (P24E8/P77C4/P50G1) -katE-sodB-rrnBT acid-resistant expression plasmid pACYC184- (P24E8/P77C4/P50G1) -katE-sodB-rrnBT.
FIG. 7 shows the construction of hdeB-sodB-katE three-gene acid-resistant expression cassette expression plasmid pACYC184P-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT.
FIG. 8 shows the construction of gadE-sodB-katE-hdeB four-gene antacid expression cassette expression plasmid pACYC184-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT.
FIG. 9 shows the results of acid pressure growth test of the Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT antacid expression cassette.
FIG. 10 shows the results of acid stress growth test of the Pasr mutant (24E8/77C4/50G1) -sodB-rrnBT acid-resistant expression cassette.
FIG. 11 shows the results of acid stress growth test of the Pasr mutant (24E8/77C4/50G1) -katE-rrnBT antacid expression cassette.
FIG. 12 shows the results of acid stress growth test of the Pasr mutant (24E8/77C4/50G1) -hdeB-rrnBT acid-resistant expression cassette.
FIG. 13 shows the results of acid pressure growth test of the Pasr mutant (24E8/77C4/50G1) -katE-sodB-rrnBT antacid expression cassette.
FIG. 14 shows the results of acid stress growth test of partially recombinant strain of sodB-katE-hdeB three-gene acid-resistant expression cassette.
FIG. 15 shows the results of acid stress growth test of gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette partial recombinant strain.
Detailed Description
The invention will now be further illustrated by way of the following examples, without thereby limiting the invention to the described examples.
An acid-resistant expression cassette, which consists of more than one promoter, more than one stress-resistant gene and more than one terminator, wherein the stress-resistant gene is selected from a gadE gene, a sodB gene, a katE gene and a hdeB gene, and an acid-response asr promoter library is constructed; the gadE gene codes an amino acid sequence shown as SEQ ID NO. 1; the sodB gene codes an amino acid sequence shown as SEQ ID NO. 2; the katE gene encodes an amino acid sequence shown as SEQ ID NO. 3; the hdeB gene encodes an amino acid sequence shown in SEQ ID NO. 4.
Further, the promoter is a pH-responsive promoter; the pH response promoter is asr promoter or mutant thereof, and the gene code is shown in SEQ ID NO. 10-59; the terminator is rrnB terminator; the nucleotide sequence of the rrnB terminator is shown in SEQ ID NO 9.
Further, it consists of asr promoter mutant selected from SEQ ID NO. 11-13, gadE gene, and rrnB terminator from 5 'to 3'; the nucleotide sequence is shown in any one of SEQ ID NO 60-62.
Further, from 5 'to 3', consisting of asr promoter mutant selected from SEQ ID NO:11-13, sodB gene, and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 63-65.
Further, from 5 'to 3', consisting of asr promoter mutant selected from SEQ ID NO:11-13, katE gene, and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 66-68.
Further, from 5 'to 3', consisting of asr promoter mutant selected from SEQ ID NO. 11-13, hdeB gene, and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 13-15.
Further, from 5 'to 3', consisting of asr promoter mutant selected from SEQ ID NO:11-13, sodB gene, katE gene, and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 69-71.
An acid-resistant expression cassette consisting of three parts, a first part comprising from 5 'to 3' an asr promoter mutant, a sodB gene, and a rrnB terminator, a second part comprising from 5 'to 3' a asr promoter mutant, a katE gene, and a rrnB terminator, and a third part comprising from 5 'to 3' a asr promoter mutant, a hdeB gene, and a rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 75 and 76.
An acid-resistant expression cassette consisting of four parts, a first part comprising from 5 'to 3' asr promoter mutant, gadE gene and rrnB terminator, a second part comprising from 5 'to 3' asr promoter mutant, sodB gene and rrnB terminator, a third part comprising from 5 'to 3' asr promoter mutant, katE gene and rrnB terminator, and a fourth part comprising from 5 'to 3' asr promoter mutant, hdeB gene and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 77-81.
An invention also includes an expression construct of the expression cassette and a recombinant host cell of the expression cassette. The host cell of the recombinant host cell is a prokaryotic cell, preferably a bacterial cell, more preferably E.coli.
Use of an antacid expression cassette comprising the steps of:
(a) introducing the expression cassette of any one of SEQ ID NOs.78-81 of claim 9 into an organic acid-producing microorganism;
(b) fermenting the microorganism;
(c) the resulting organic acid is harvested.
In the above application, the organic acid includes amino acid, succinic acid, citric acid and lactic acid; the amino acid includes lysine, threonine, tryptophan or glutamic acid.
In the above application, the microorganism is a prokaryotic microorganism, preferably a bacterium, and more preferably Escherichia coli.
A method for high throughput screening of stress-resistant expression cassettes for microbial fermentation, said method comprising the steps of:
(a) introducing an antacid expression cassette (gadE-sodB-katE-hdeB) to be screened into the microorganism;
(b) evaluating the growth rate of the microorganisms under the stress pressure environment by using a high-throughput growth tester;
the is BioscreenC;
(c) evaluating the fermentation performance of the microorganism in a stress pressure environment by using a fermentation tank;
(d) identifying an expression cassette capable of increasing said growth rate and/or said fermentation performance as a stress resistant expression cassette for use in a microbial fermentation.
Example 1: regulatory elements for construction of asr promoter mutation library
In this example, the asr promoter mutant library was constructed as shown in FIG. 1 by randomizing the 9bp sequence between-35 and-10 of the asr promoter in the pACYC184-Pasr-mCherry-rrnBT plasmid.
PCR amplification was performed using pACYC184-Pasr-mCherry-rrnBT as a template with the following forward and reverse primers according to the conventional method to obtain polynucleotide fragment 1 for Gibson assembly: upstream primer 5'-CTGCTGGCTACCCTGTGGAA-3' (Pasrori lib-mCherrygi1-for, SEQ ID NO:82) and downstream primer 5 '-ACACTTCCGTGAGTGGTTGGTTTCAGNNNNNNNNGATATGTACAAACGCTG-3' (Pasrori lib-mCherrygi1-rev, SEQ ID NO: 83). PCR was performed using pACYC184-Pasr-mCherry-rrnBT as a template and the following forward and reverse primers according to a conventional method to obtain polynucleotide fragment 2 for Gibson assembly: an upstream primer 5'-GAAACCAACCACTCACGGAAGTCTGCCATTCCCAGGATATAGTTATTTCAACGGCCCC-3' (Pasrori lib-mCherrygi2-for, SEQ ID NO:84) and a downstream primer 5'-TTCCACAGGGTAGCCAGCAGCATC-3' (Pasrori lib-mCherrygi1-rev, SEQ ID NO: 85). Q5 polymerase from New England Biolab was used for PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 60sec, 72 ℃ 120 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniprep kit of Tiangen company. Adding the separated and recovered polynucleotide fragments into Gibson assembly mixed liquor for connection, wherein the reaction conditions are as follows: 60min at 50 ℃. The ligation products were transformed into E.coli MG1655 competent cells, the transformed cells were plated on LB (Luria-Bertani medium) plates supplemented with 34. mu.g/mL chloramphenicol to screen positive clones, plasmids were extracted and sequenced, which indicated that the cloned pACYC184-Pasr library-mCherry-rrnBT sequence was correct.
The promoter mutants were screened for characterization by fluorescence values of growth under stress pressure. The asr promoter mutants with different intensities were selected as elements for regulating the subsequent expression cassette, and the sequence and intensity information are shown in table 1. The wild type and all promoter mutant sequences are SEQ ID NO 10-59, and the strength information is shown in Table 2.
Asr promoter mutant sequence and strength selected in Table 1
Figure BDA0003421417670000091
Figure BDA0003421417670000101
Figure BDA0003421417670000111
Example 2: constructing acid-resistant expression cassettes of gadE, sodB, katE, hdeB and katE-sodB
2.1 construction of pACYC184 expression vector containing the Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT acid-resistant expression cassette
The procedure for constructing the Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT expression vector pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT used in the examples of this application is shown in FIG. 2.
The gene gadE polynucleotide fragment was obtained by first extracting the E.coli MG1655 genome using a kit (Tiangen, DP302) as a template and performing PCR amplification using the following forward and reverse primers according to a conventional method: an upstream primer 5'-CAAATTGAGGGTATGACAATGATTTTTCTCATGACGAAAG-3' (gadE-for, SEQ ID NO:86), and a downstream primer 5'-CCTTTCGTTTTATTTGATGCCTCTCGAGCTAAAAATAAGATGTGATAC-3' (gadE-rev, SEQ ID NO: 87). Q5 polymerase (New Englend Biolab, M0491) was used for the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 30 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniprep kit (Tiangen, DP 209).
PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-GACCGGGTCGAATTTGCTTTCG-3' (Pasr-comgi1-for, SEQ ID NO:94), and a downstream primer 5'-TGTCATACCCTCAATTTGT-3' (Pasr-comgi1-rev, SEQ ID NO: 95). PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-CTCGAGAGGCATCAAATAAAACG-3' (Pasr-comgi2-for, SEQ ID NO:96), and a downstream primer 5'-GAAATTCGAAAGCAAATTCGACC-3' (Pasr-comgi2-rev, SEQ ID NO: 97). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
And adding the obtained PCR product into Gibson assembly mixed liquor for connection, transforming the connection product into E.coli MG1655 competent cells, coating the transformed cells on an LB (Luria-Bertani culture medium) plate added with 34 mu G/mL chloramphenicol for screening positive clones, extracting plasmids, sequencing the plasmids, and indicating that the cloned pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -gadE-rrnBT has correct sequence.
2.2 construction of pACYC184 expression vector containing the Pasr mutant (P24E8/P77C4/P50G1) -sodB-rrnBT acid-resistant expression cassette
The construction process of the Pasr mutant (P24E8/P77C4/P50G1) -SOdB-rrnBT expression vector pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -SOdB-rrnBT used in the examples of this application is shown in FIG. 3.
Taking an Escherichia coli MG1655 genome as a template, and carrying out PCR amplification by using a forward primer and a reverse primer according to a conventional method to obtain a gene sodB polynucleotide fragment: an upstream primer 5'-CAAATTGAGGGTATGACAATGTCATTCGAATTACCTG-3' (sodB-for, SEQ ID NO:88), and a downstream primer 5'-CCTTTCGTTTTATTTGATGCCTCTCGAGTTATGCAGCGAGATTTTTC-3' (sodB-rev, SEQ ID NO: 89). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 30 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-GACCGGGTCGAATTTGCTTTCG-3' (Pasr-comgi1-for, SEQ ID NO:94), and a downstream primer 5'-TGTCATACCCTCAATTTGT-3' (Pasr-comgi1-rev, SEQ ID NO: 95). PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-CTCGAGAGGCATCAAATAAAACG-3' (Pasr-comgi2-for, SEQ ID NO:96), and a downstream primer 5'-GAAATTCGAAAGCAAATTCGACC-3' (Pasr-comgi2-rev, SEQ ID NO: 97). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
And adding the obtained PCR product into a Gibson assembly mixed solution for connection, transforming the connection product into E.coli MG1655 competent cells, coating the transformed cells on an LB (Luria-Bertani culture medium) plate added with 34 mu G/mL chloramphenicol for screening positive clones, extracting plasmids, sequencing the plasmids, and indicating that the cloned pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -sodB-rrnBT has correct sequence.
2.3 construction of pACYC184 expression vector containing the Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT acid-resistant expression cassette
The procedure for constructing the Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT expression vector pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT used in this example is shown in FIG. 4.
The gene katE polynucleotide fragment was obtained by PCR amplification using the E.coli MG1655 genome as a template and the following forward primer and reverse primer according to the conventional method: an upstream primer 5'-CAAATTGAGGGTATGACAATGTCGCAACATAACGAAAAG-3' (katE-for, SEQ ID NO:90), and a downstream primer 5'-CCTTTCGTTTTATTTGATGCCTCTCGAGTCAGGCAGGAATTTTGTC-3' (katE-rev, SEQ ID NO: 91). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-GACCGGGTCGAATTTGCTTTCG-3' (Pasr-comgi1-for, SEQ ID NO:94), and a downstream primer 5'-TGTCATACCCTCAATTTGT-3' (Pasr-comgi1-rev, SEQ ID NO: 95). PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-CTCGAGAGGCATCAAATAAAACG-3' (Pasr-comgi2-for, SEQ ID NO:96), and a downstream primer 5'-GAAATTCGAAAGCAAATTCGACC-3' (Pasr-comgi2-rev, SEQ ID NO: 97). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
The obtained PCR product is added into Gibson assembly mixed liquid for connection, the connection product is transformed into E.coli MG1655 competent cells, the transformed cells are coated on LB (Luria-Bertani culture medium) plates added with 34 ug/mL chloramphenicol for screening positive clones, plasmids are extracted and sequenced, and the sequencing result shows that the cloned pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT has correct sequence.
2.4 construction of pACYC184 expression vector containing the Pasr mutant (P24E8/P77C4/P50G1) -hdeB-rrnBT acid-resistant expression cassette
The construction of the Pasr mutant (P24E8/P77C4/P50G1) -hdeB-rrnBT expression vector pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -hdeB-rrnBT used in the examples of this application is shown in FIG. 5.
The gene hdeB polynucleotide fragment is obtained by PCR amplification by using Escherichia coli MG1655 genome as a template and using the following forward primer and reverse primer according to a conventional method: an upstream primer 5'-CCAAATTGAGGGTATGACAATGAATATTTCATCTCTCCG-3' (hdeB-for, SEQ ID NO:92), and a downstream primer 5'-CCTTTCGTTTTATTTGATGCCTCTCGAGTTAATTCGGCAAGTCATTAG-3' (hdeB-rev, SEQ ID NO: 93). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 30 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-GACCGGGTCGAATTTGCTTTCG-3' (Pasr-comgi1-for, SEQ ID NO:94), and a downstream primer 5'-TGTCATACCCTCAATTTGT-3' (Pasr-comgi1-rev, SEQ ID NO: 95). PCR was performed using pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -mCherry-rrnBT as a template with the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment: an upstream primer 5'-CTCGAGAGGCATCAAATAAAACG-3' (Pasr-comgi2-for, SEQ ID NO:96), and a downstream primer 5'-GAAATTCGAAAGCAAATTCGACC-3' (Pasr-comgi2-rev, SEQ ID NO: 97). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
And adding the obtained PCR product into Gibson assembly mixed liquor for connection, transforming the connection product into E.coli MG1655 competent cells, coating the transformed cells on an LB (Luria-Bertani culture medium) plate added with 34 mu G/mL chloramphenicol for screening positive clones, extracting plasmids, sequencing the plasmids, and indicating that the cloned pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -hdeB-rrnBT has correct sequence.
2.5 construction of pACYC184 expression vector for Pasr mutant (P24E8/P77C4/P50G1) -katE-sodB-rrnBT acid-resistant expression cassette
The procedure for constructing the mutant Pasr (P24E8/P77C4/P50G1) -katE-sodB-rrnBT expression vector pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-sodB-rrnBT used in the examples of this application is shown in FIG. 6.
Using the pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -sodB-rrnBT constructed in example 2 as a template, PCR was performed using the following forward and reverse primers to obtain a Gibson assembly polynucleotide fragment RBS-sodBgi: an upstream primer 5'-CGCAGGTGTGGTTATGCTACTAGAGACGGCCCCGCAGTGGGGTT-3' (RBS-sodBgi-for, SEQ ID NO:98), and a downstream primer 5'-GCCTTTCGTTTTATTTGATGCCTCTC-3' (RBS-sodBgi-rev, SEQ ID NO: 99). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 30sec, 72 ℃ 30 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
Using the pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT constructed in example 2 as a template, PCR was performed using the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment katE-sodBgi 1: an upstream primer 5'-CGCTAGCGGAGTGTATACTGGCTTACTATGTT-3' (katE-sodBgi1-for, SEQ ID NO:100), and a downstream primer 5'-CTCTAGTAGCATAACCACACCTGCGTCAGGCAGGAATTTTGTCAA-3' (katE-sodBgi1-rev, SEQ ID NO: 101). Using the pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-rrnBT constructed in example 2 as a template, PCR was performed using the following forward and reverse primers to obtain a Gibson assembled polynucleotide fragment katE-sodBgi 2: an upstream primer 5'-CTCGAGAGGCATCAAATAAAACG-3' (katE-sodBgi2-for, SEQ ID NO:102), and a downstream primer 5'-TAAGCCAGTATACACTCCGCTAGC-3' (katE-sodBgi2-rev, SEQ ID NO: 103). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 60sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
The obtained PCR product is added into Gibson assembly mixed liquid for connection, the connection product is transformed into E.coli MG1655 competent cells, the transformed cells are coated on LB (Luria-Bertani culture medium) plates added with 34 ug/mL chloramphenicol for screening positive clones, plasmids are extracted and sequenced, and the sequencing result shows that the cloned pACYC184-Pasr mutant (P24E8/P77C4/P50G1) -katE-sodB-rrnBT sequence is correct.
Example 3: SodB-katE-hdeB three-gene acid-resistant expression cassette
3.1 construction of pACYC184 expression vector containing triple-Gene Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT acid-resistant expression cassette
The construction process of the Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT used in the examples of this application is shown in FIG. 7.
Using pACYC184-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT constructed in example 2 as a template, BsaI-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-BsaI polynucleotide fragment was obtained by PCR amplification according to the conventional method using the following forward primer and reverse primer: upstream primer 5-GGTCTCAGTCCATTCAGCGTTTGTACATAT-3' (BsaI-f2-f3-for, SEQ ID NO:106, underlined bases being the recognition site for the restriction enzyme BsaI) and the downstream primer 5-GGTCTCAAGATTAGATATGACGACAGGAAG-3' (BsaI-f2-f3-rev, SEQ ID NO:107, underlined bases as restriction enzyme BsaI recognition sites). Using the pACYC184-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE constructed in example 2 as a template, a BsaI-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-BsaI polynucleotide fragment was obtained by PCR amplification according to the conventional method using the following forward primer and reverse primer: upstream primer 5-GGTCTCAATCTATTCAGCGTTTGTACATAT-3' (BsaI-f3-f4-for, SEQ ID NO:108, underlined bases being the recognition site for the restriction enzyme BsaI) and the downstream primer 5-GGTCTCAACTCTAGATATGACGACAGGAAG-3' (BsaI-f3-f4-rev, SEQ ID NO:109, underlined bases as restriction enzyme BsaI recognition sites). Using the pACYC184-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT constructed in example 2 as a template, a BsaI-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-BsaI polynucleotide fragment was obtained by PCR amplification according to the conventional method using the following forward primer and reverse primer: upstream primer 5-GGTCTCAGCTTATTCAGCGTTTGTACATAT-3' (BsaI-f4-f5-for, SEQ ID NO:110, underlined bases being the recognition site for the restriction enzyme BsaI) and the downstream primer 5-GGTCTCAAAGCTAGATATGACGACAGGAAG-3' (BsaI-f4-f5-rev, SEQ ID NO:111, underlined bases as restriction enzyme BsaI recognition sites). In embodiment 1The constructed pACYC184-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT was used as a template, and the BsaI-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-BsaI polynucleotide fragment was obtained by PCR amplification according to the conventional method using the following forward primer and reverse primer: upstream primer 5-GGTCTCAGCTTATTCAGCGTTTGTACATAT-3' (BsaI-f4-f5-for, SEQ ID NO:110, underlined bases being the recognition site for the restriction enzyme BsaI) and the downstream primer 5-GGTCTCAACTCTAGATATGACGACAGGAAG-3' (BsaI-f4-f5-rev, SEQ ID NO:111, underlined bases as restriction enzyme BsaI recognition sites). Q5 polymerase was used in the PCR reaction under the following conditions: 30sec at 98 ℃; 30 cycles of 98 ℃ 10sec, 60 ℃ 60sec, 72 ℃ 60 sec; 72 ℃ for 2 min. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and amplifying a correct band which is consistent with the expectation by the result of PCR. Then, gel separation and recovery are carried out by using a high-purity DNA fragment miniextraction kit.
The isolated and recovered polynucleotide fragment BsaI-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT-BsaI, BsaI-Pasr mutant (24E8/10G6/77C4/50G1) -hdeB-rrnBT-BsaI, BsaI-Pasr mutant (24E8/10G6/77C4/50G1) -sodB-rrnBsaI and BsaI-Pasr mutant BT 24E8/10G6/77C4/50G1) -katE-rrnBsaI was treated with T5 Mix polymerase (Tsingke, TSE005) by PCR at the 3' end of the recovered fragment with A, under the following PCR conditions: 10min at 72 ℃. After the reaction was completed, the DNA was purified and recovered using an ultrathin DNA product purification kit (Tiangen, DP 203-02). Purification of Polynucleotide fragments Using pMDTM19T cloning kit (TaKaRa, 6013) was ligated with pMD19T vector according to the kit instructions. The ligation product was transformed into E.coli DH 5. alpha. competent cells, the transformed cells were plated on LB (Luria-Bertani medium) plates supplemented with 50. mu.g/mL ampicillin to select positive clones, plasmids were extracted and sequenced, and the sequencing results showed that the cloned pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-nBT and pMD 19-Pasr mutant (P24E 3729/P8/P10G 4642/P50G 4) -norrB-contain the correct sequences.
pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT, and pACYC184-ccdB-rrnBT were digested and ligated with restriction enzymes Bsa (New Engled Biolab, R3733) and T4 ligase (New Engled Biolab, M0202), and the reaction was carried out in a PCR apparatus under the following conditions: 10min at 37 ℃, 7min at 16 ℃, 5min at 50 ℃ and 5min at 80 ℃. The ligation products were transformed into e.coli MG1655 competent cells, the transformed cells were plated on LB (Luria-Bertani medium) plates supplemented with 34 μ g/mL chloramphenicol to screen positive clones, plasmids were extracted, and sequencing was performed thereon to identify promoter-mutant combinations.
Example 4: construction of gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette
4.1 construction of four-Gene Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -SOdB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT antacid expression cassette
The construction process of the Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT used in the examples of this application is shown in FIG. 8.
pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -gadE-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -sodB-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -katE-rrnBT, pMD19T-Pasr mutant (P24E8/P10G6/P77C4/P50G1) -hdeB-rrnBT and pACYC184-ccdB-rrnBT were digested with restriction endonuclease Bsa I and T4 ligase, ligated, and reacted in a PCR apparatus under the following conditions: 10min at 37 ℃, 7min at 16 ℃, 5min at 50 ℃ and 5min at 80 ℃. The ligation products were transformed into e.coli MG1655 competent cells, the transformed cells were plated on LB (Luria-Bertani medium) plates supplemented with 34 μ g/mL chloramphenicol to screen positive clones, plasmids were extracted, and sequencing was performed thereon to identify promoter-mutant combinations.
Example 5 Effect of the acid-resistant expression cassette on acid-stressed growth of Escherichia coli MG1655
5.1 the acid-fast expression cassette plasmid-containing strain, the control plasmid-containing strain and Escherichia coli MG1655 obtained in example 2-4 were inoculated into LBG medium at 37 ℃ overnight at 250rpm, and cultured at the initial OD6000.05 was transferred to 300. mu.L of fresh LBG-pH5.0 medium and a high throughput Growth assay was performed using a fully automated Growth curve analyzer Bioscreen C (Oy Growth cultures Ab Ltd, Finland) and 100-well plates (Honeycompb Plate, 2 plates were used simultaneously).
The strains obtained in example 2-4 were inoculated in LB medium at 37 ℃ overnight at 250rpm, mixed with 0.8mL of the inoculum and 0.2mL of 60% glycerol, and frozen in a freezer at-80 ℃. The strain corresponding to the expression plasmid transferred into the monogenic acid-resistant expression cassette or katE-sodB acid-resistant expression cassette is named as the name of the acid-resistant expression cassette. The strains transferred into the three-gene and four-gene acid-resistant expression cassettes respectively contain 64 and 256 unique promoter combinations, a part of single colonies are picked for sequencing, 31 and 244 unique combinations are respectively obtained in the hdeB-sodB-katE and gadE-hdeB-sodB-katE acid-resistant expression cassettes after identification, and the corresponding strains are respectively named as hsk and ghsk.
The above-frozen strain containing the acid-fast expression cassette plasmid, the strain containing the control plasmid, and E.coli MG1655 were restored to LB solid medium plates (for E.coli MG1655, no antibiotic was added; for the strain containing the acid-fast expression cassette plasmid, the control plasmid, chloramphenicol at 34. mu.g/mL; the same applies hereinafter), and cultured overnight at 37 ℃. The cells were inoculated into LB medium supplemented with 2% glucose, respectively, and cultured overnight at 37 ℃ and 250 rpm. At the initial OD6000.05 to 300. mu.L of fresh LBG-pH5.0 medium, a high throughput Growth assay was performed using a fully automated Growth curve analyzer Bioscreen C (Oy Growth cultures Ab Ltd, Finland) and 100-well plates (Honeycompb Plate, 2 plates were used simultaneously).
As a result:
1. for the gadE acid-resistant expression cassette, the acid stress growth test results are shown in FIG. 9.
Growth of the respective strains after 24 hours of growth in LBG MediumLong (initial time measured by Bioscreen C and OD at 24 hours time)600) As shown in table 2.
TABLE 2 acid stress growth assay for strains containing the gadE acid-resistant expression cassette
Figure BDA0003421417670000181
(1) The strain P24E8-gadE containing the gadE acid-resistant expression cassette was not increased, whereas P77C4-gadE and P50G1-gadE, grown 24 hours, were increased by 4.1% and 1.8% in LBG-pH5.0 medium, respectively, compared to the control strain MG 1655.
(2) The effect of the gadE acid-resistant expression cassette on improving acid pressure growth tolerance is not obvious, and gadE acid-resistant expression cassettes with different expression strengths bring about improvement of acid pressure growth tolerance of strains in different degrees, wherein the improvement of the acid-resistant expression cassette P77C4-gadE and the improvement of the growth of a control strain E.coli MG1655 in an LBG medium at 24 hours is improved by 3.8%.
3. For the sodB acid-fast expression cassette, the acid pressure growth test results are shown in fig. 10.
Growth of each strain after 24 hours of growth in LBG Medium (OD of initial and 24 hours measured by Bioscreen C)600) As shown in table 3.
TABLE 3 acid stress growth test of strains containing the sodB acid-resistant expression cassette
Figure BDA0003421417670000191
(1) The strains P24E8-sodB, P77C4-sodB and P50G1-sodB containing the sodB acid-fast expression cassette did not increase in LBG-pH5.0 medium for 24 hours compared to the control strain MG 1655.
(2) The lag phase of strains P24E8-sodB, P77C4-sodB and P50G1-sodB containing the sodB acid-fast expression cassette was significantly increased when grown in LBG-pH5.0 medium.
4. For the katE acid-fast expression cassette, the acid stress growth test results are shown in FIG. 11.
Growth of each strain after 24 hours of growth in LBG Medium (Bioscreen)OD of C measured initial time and 24 hours time600) As shown in table 4.
TABLE 4 acid stress growth assay for strains containing katE acid-resistant expression cassettes
Figure BDA0003421417670000192
(1) The strains P24E8-katE, P77C4-katE and P50G1-katE containing the katE acid-fast expression cassette were grown in LBG-pH5.0 medium for 24 hours by 4.8%, 32.8% and 10.4%, respectively, compared to the control strain MG 1655.
(2) The katE acid-resistant expression cassette was able to improve the acid stress growth tolerance, and the katE acid-resistant expression cassettes of different expression strengths gave rise to different degrees of acid stress growth tolerance of the strains, with the acid-resistant expression cassette P77C4-katE being the most elevated, with a 32.8% improvement in growth of the control strain e.coli MG1655 at 24 hours time on LBG medium.
5. For the hdeB antacid expression cassette, the acid stress growth test results are shown in FIG. 12.
Growth of each strain after 24 hours of growth in LBG Medium (OD of initial and 24 hours measured by Bioscreen C)600) As shown in table 5.
TABLE 5 acid stress growth assay for strains containing hdeB acid-resistant expression cassettes
Figure BDA0003421417670000201
(1) The strain P24E8-hdeB containing the hdeB antacid expression cassette was not increased, whereas P77C4-hdeB and P50G1-hdeB, grown 24 hours, were increased by 4.5% and 33.7% in LBG-pH5.0 medium, respectively, compared to the control strain MG 1655.
(2) The hdeB acid-resistant expression cassette can improve the acid stress growth tolerance, and the hdeB acid-resistant expression cassettes with different expression strengths bring about the improvement of the acid stress growth tolerance of the strains in different degrees, wherein the improvement of the acid-resistant expression cassette P50G1-hdeB is the largest, and the improvement of the growth of a control strain E.coli MG1655 in LBG medium at 24 hours is 33.7 percent.
6. For the katE-sodB acid-fast expression cassette, the results of the acid stress growth assay are shown in FIG. 13.
Growth of each strain after 24 hours of growth in LBG Medium (OD of initial and 24 hours measured by Bioscreen C)600) As shown in table 6.
TABLE 6 acid pressure growth assay for strains containing the katE-sodB acid-resistant expression cassette
Figure BDA0003421417670000202
(1) The strains P24E8-katE-sodB, P77C4-katE-sodB and P50G1-katE-sodB containing the katE-sodB acid-resistant expression cassette were grown in LBG-pH5.0 medium for 24 hours at 5.4%, 20.8% and 12.0% respectively, compared with the control strain MG 1655.
(2) The katE-sodB acid-resistant expression cassette can improve the acid pressure growth tolerance, and the katE-sodB acid-resistant expression cassettes with different expression intensities bring about the improvement of the acid pressure growth tolerance of the strains in different degrees, wherein the improvement of the acid-resistant expression cassette P77C4-katE-sodB is the largest, and the improvement of the growth of a control strain E.coli MG1655 in LBG culture medium at the 24-hour time is 20.8 percent.
7. The results of the acid stress growth test for the sodB-katE-hdeB three-gene acid-resistant expression cassette are shown in FIG. 14.
There are 64 combinations of the sodB-katE-hdeB three-gene acid-resistant expression cassette, and 31 combinations are identified, and the strains are named by hsk. Growth of the Strain with Pre-viability 2 after 24 hours of growth in LBG Medium (OD of initial time and 24 hours measured by Bioscreen C)600) As shown in table 7.
TABLE 7 acid pressure growth test of the first 2 strains of sodB-katE-hdeB three-Gene acid-resistant expression cassette viability
Figure BDA0003421417670000211
(1) The strain containing the sodB-katE-hdeB three-gene acid-resistant expression cassette is obviously improved in LBG-pH5.0 culture medium compared with a control strain MG1655 after 24 hours of growth, the strain with the first viability 2 is respectively improved by 56.2 percent and 45.0 percent, wherein the improvement range of hsk2A10 is the largest.
8. The results of the acid stress growth test for the gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette are shown in FIG. 15.
There were 256 combinations of gadE-sodB-katE-hdeB four-gene acid-resistant expression cassettes, and a total of 244 combinations were identified, and the strains were named ghsk. Growth of the Strain with Pre-viability 2 after 24 hours of growth in LBG Medium (OD of initial time and 24 hours measured by Bioscreen C)600) As shown in table 10.
TABLE 10 GAdE-sodB-katE-hdeB four Gene acid-resistant expression cassette viability Pre-5 Strain acid pressure growth test
Figure BDA0003421417670000212
1) The strain containing the gadE-sodB-katE-hdeB four-gene antacid expression cassette is obviously improved in LBG-pH5.0 culture medium compared with the control strain MG1655 after 24 hours of growth, and the strains with the first viability 2 are respectively improved by 49.5 percent and 47.0 percent, wherein the ghsk 4F2 is improved most obviously.
Example 6: effect of acid-resistant expression cassette on lysine fermentation
4 plasmids of gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette expression plasmid obtained in example 4 (pACYC184-P77C4-gadE-rrnBT-P10G6-sodB-rrnBT-P50G1-katE-rrnBT-P24E8-hdeB-rrnBT (ghsk 3C5), CYC184-P77C4-gadE-rrnBT-P77C4-sodB-rrnBT-P24E8-katE-rrnBT-P77C4-hdeB-rrnBT (gk 10C3), pACYC184-P10G 6-gadE-nbt-P10G 2-sorrnBT-24E-24-gadE-24-prtBT-24G 35rrnBT 24G 9-9G 2-sornB-sornBT-24E 24-prgBstnBT 24G 26), pACYC 184-prcBrrnBT 24G 35nrnBT 24G 3512-9-prgE 11-prgE 11-prgE 28-prgE 11-prgE 28-prrH 28-prrBgE-prgE-prrBgSbE 11-prrBgSbE 2-prrBgSbE-prgSbE 28-prgSbE-prbE 28-prgSbE 28-prbE 28-prbE 28-prbE 28-9-prbE 28-prbE 28-prbE-E-prbE The strain MG1655 (the strain is based on the strain MG1655 and is transformed into competent cells according to the method disclosed in Chinese patent application CN103773745A to obtain the lysine-producing strain SCEcL3), and positive clone is obtained by colony PCR and plasmid sequencing identification. Inoculating to LB culture medium at 37 deg.C, culturing overnight at 250rpm, mixing with 0.8mL bacterial liquid and 0.2mL 60% glycerol, and freezing in-80 deg.C refrigerator. The corresponding strains were named: parent lysine-producing strain SC, acid-fast expression cassette strain SC ghsk containing gadE-sodB-katE-hdeB four genes, and plasmid code name is added.
The above-frozen lysine-producing strains containing the acid-resistant expression cassette plasmid and the parent lysine-producing strains were returned to LB solid medium plates (34. mu.g/mL chloramphenicol was added to the lysine-producing strain containing the acid-resistant expression cassette plasmid), and cultured overnight at 37 ℃. The strains were inoculated into 2mL seed medium at 37 ℃ overnight at 200 rpm. Then, the cells were transferred to 100mL Erlenmeyer flasks containing seed medium and cultured overnight at 37 ℃ and 200 rpm. The cells were transferred to a fermentation medium at a rate of 15% in a total volume of 500mL, and a high throughput acid pressure fermentation test was performed using a 1.3-L fermentor.
The seed culture medium is as follows: 3g/L of sucrose, 5g/L of yeast extract, 7g/L of casein, 5g/L of ammonium sulfate, 5g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.012g/L of ferric sulfate, 0.012g/L of manganese sulfate, 5g/L of sodium glutamate, 0.3g/L of L-threonine, 0.3g/L of L-methionine and 0.3g/L of pyruvic acid.
The fermentation medium was as follows: 30g/L of glucose, 10g/L of ammonium sulfate, 0.6g/L of phosphoric acid, 0.5g/L of potassium chloride, 2.2g/L of betaine, 2g/L of magnesium sulfate, 0.032g/L of manganese sulfate, 0.03g/L of ferrous sulfate, 0.325g/L of corn steep liquor, 0.032g/L of ferrous sulfate, 0.25g/L of L-threonine, 6.8mg/L of copper sulfate, 7.65mg/L of zinc sulfate, 5.6mg/L of thiamine and 0.5mL/L of 3% defoaming agent. Adjusting pH of the culture medium to 7.0 with ammonia water, wherein glucose and phosphoric acid are sterilized separately, and manganese sulfate, ferrous sulfate, L-threonine, copper sulfate, zinc sulfate and thiamine are sterilized separately and added into the culture medium. The initial pH of the fermentation medium was 7.0 in the fermentation test, the pH was lowered to 6.0 after about 6 hours of fermentation, and then the pH was maintained at 6.0 by feeding ammonia water and fermented in a 1.3-L fermenter for a total of 48 hours. The fermentation broth was analyzed for lysine-HCl content using a biosensor analyzer SBA-40D (institute of biological research, academy of sciences, Shandong province).
As a result:
for the gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette, there were 4 in total, and the results after 48 hours of fermentation are shown in Table 11.
TABLE 11 acid pressure fermentation test of lysine-producing strains containing gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette
Figure BDA0003421417670000231
(1) For acid-resistant expression cassette containing gadE-sodB-katE-hdeB four genes under the fermentation conditions of the fermentation medium and pH 6.0
The lysine-producing strain SC ghsk 3C5 of P77C4-gadE-rrnBT-P10G6-sodB-rrnBT-P50G1-katE-rrnBT-P24E8-hdeB-rrnBT has a lysine-HCl yield of 114.9% of that of the parent lysine-producing strain SC at the pH value of 6.0 in 48 hours, which is higher than that of the parent lysine-producing strain SC. The saccharic acid conversion rate in 48 hours is 114.8 percent of the parent lysine-producing strain P at the pH value of 6.0, is obviously higher than that of the parent lysine-producing strain P, and is 105.4 percent of the parent lysine-producing strain P at the pH value of 6.8.
(2) For acid-resistant expression cassette containing gadE-sodB-katE-hdeB four genes under the fermentation conditions of the fermentation medium and pH 6.0
The lysine-producing strain SC ghsk 10C3 of P77C4-gadE-rrnBT-P77C4-hdeB-rrnBT-P77C4-sodB-rrnBT-P24E8-katE-rrnBT has the lysine-HCl yield of 101.1 percent of the parent lysine-producing strain SC at the pH value of 6.0 in 48 hours, and has no obvious difference with the parent lysine-producing strain SC. The saccharic acid conversion rate in 48 hours is 94.4% of the parent lysine-producing strain SC at pH 6.0, is lower than the parent lysine-producing strain SC, and is 86.7% of the parent lysine-producing strain SC at pH 6.8.
(3) For acid-resistant expression cassette containing gadE-sodB-katE-hdeB four genes under the fermentation conditions of the fermentation medium and pH 6.0
The lysine-producing strain SC ghsk 5F5 of P10G6-gadE-rrnBT-P10G6-sodB-rrnBT-P24E8-katE-rrnBT-P24E8-hdeB-rrnBT has the lysine-HCl yield of 112.7 percent of the parent lysine-producing strain SC at the pH value of 6.0 in 48 hours, and has no obvious difference with the parent lysine-producing strain SC. The saccharic acid conversion rate in 48 hours is 105.4% of the parent lysine-producing strain SC at pH 6.0, is lower than that of the parent lysine-producing strain SC, and is 96.8% of that of the parent lysine-producing strain SC at pH 6.8.
(4) For acid-resistant expression cassette containing gadE-sodB-katE-hdeB four genes under the fermentation conditions of the fermentation medium and pH 6.0
The lysine-producing strain SC ghsk 6H10 of P24E8-gadE-rrnBT-P50G1-sodB-rrnBT-P50G1-katE-rrnBT-P77C4-hdeB-rrnBT has the lysine-HCl yield of 102.8 percent of the parent lysine-producing strain SC at the pH value of 6.0 in 48 hours, and has no obvious difference with the parent lysine-producing strain SC. The saccharic acid conversion rate in 48 hours is 95.5% of that of the parent lysine-producing strain SC at pH 6.0, is lower than that of the parent lysine-producing strain SC, and is 87.6% of that of the parent lysine-producing strain SC at pH 6.8.
Although all of the acid-resistant expression cassettes constructed in the examples can improve the acid resistance of E.coli after transformation of E.coli, it is surprising that different combinations of different promoters, stress-resistant genes and terminators have significantly different effects on E.coli fermentation. For the fermentation production of lysine, the lysine-producing strain SC ghsk 3C5 containing the gadE-sodB-katE-hdeB four-gene acid-resistant expression cassette P77C4-gadE-rrnBT-P10G6-sodB-rrnBT-P50G1-katE-rrnBT-P24E8-hdeB-rrnBT obtains unexpected excellent effect.
Sequence listing
<110> university of southern China's science
<120> acid-resistant expression cassette and application thereof, and method for high-throughput screening of stress-resistant expression cassette for microbial fermentation
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<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 3
Met Ser Gln His Asn Glu Lys Asn Pro His Gln His Gln Ser Pro Leu
1 5 10 15
His Asp Ser Ser Glu Ala Lys Pro Gly Met Asp Ser Leu Ala Pro Glu
20 25 30
Asp Gly Ser His Arg Pro Ala Ala Glu Pro Thr Pro Pro Gly Ala Gln
35 40 45
Pro Thr Ala Pro Gly Ser Leu Lys Ala Pro Asp Thr Arg Asn Glu Lys
50 55 60
Leu Asn Ser Leu Glu Asp Val Arg Lys Gly Ser Glu Asn Tyr Ala Leu
65 70 75 80
Thr Thr Asn Gln Gly Val Arg Ile Ala Asp Asp Gln Asn Ser Leu Arg
85 90 95
Ala Gly Ser Arg Gly Pro Thr Leu Leu Glu Asp Phe Ile Leu Arg Glu
100 105 110
Lys Ile Thr His Phe Asp His Glu Arg Ile Pro Glu Arg Ile Val His
115 120 125
Ala Arg Gly Ser Ala Ala His Gly Tyr Phe Gln Pro Tyr Lys Ser Leu
130 135 140
Ser Asp Ile Thr Lys Ala Asp Phe Leu Ser Asp Pro Asn Lys Ile Thr
145 150 155 160
Pro Val Phe Val Arg Phe Ser Thr Val Gln Gly Gly Ala Gly Ser Ala
165 170 175
Asp Thr Val Arg Asp Ile Arg Gly Phe Ala Thr Lys Phe Tyr Thr Glu
180 185 190
Glu Gly Ile Phe Asp Leu Val Gly Asn Asn Thr Pro Ile Phe Phe Ile
195 200 205
Gln Asp Ala His Lys Phe Pro Asp Phe Val His Ala Val Lys Pro Glu
210 215 220
Pro His Trp Ala Ile Pro Gln Gly Gln Ser Ala His Asp Thr Phe Trp
225 230 235 240
Asp Tyr Val Ser Leu Gln Pro Glu Thr Leu His Asn Val Met Trp Ala
245 250 255
Met Ser Asp Arg Gly Ile Pro Arg Ser Tyr Arg Thr Met Glu Gly Phe
260 265 270
Gly Ile His Thr Phe Arg Leu Ile Asn Ala Glu Gly Lys Ala Thr Phe
275 280 285
Val Arg Phe His Trp Lys Pro Leu Ala Gly Lys Ala Ser Leu Val Trp
290 295 300
Asp Glu Ala Gln Lys Leu Thr Gly Arg Asp Pro Asp Phe His Arg Arg
305 310 315 320
Glu Leu Trp Glu Ala Ile Glu Ala Gly Asp Phe Pro Glu Tyr Glu Leu
325 330 335
Gly Phe Gln Leu Ile Pro Glu Glu Asp Glu Phe Lys Phe Asp Phe Asp
340 345 350
Leu Leu Asp Pro Thr Lys Leu Ile Pro Glu Glu Leu Val Pro Val Gln
355 360 365
Arg Val Gly Lys Met Val Leu Asn Arg Asn Pro Asp Asn Phe Phe Ala
370 375 380
Glu Asn Glu Gln Ala Ala Phe His Pro Gly His Ile Val Pro Gly Leu
385 390 395 400
Asp Phe Thr Asn Asp Pro Leu Leu Gln Gly Arg Leu Phe Ser Tyr Thr
405 410 415
Asp Thr Gln Ile Ser Arg Leu Gly Gly Pro Asn Phe His Glu Ile Pro
420 425 430
Ile Asn Arg Pro Thr Cys Pro Tyr His Asn Phe Gln Arg Asp Gly Met
435 440 445
His Arg Met Gly Ile Asp Thr Asn Pro Ala Asn Tyr Glu Pro Asn Ser
450 455 460
Ile Asn Asp Asn Trp Pro Arg Glu Thr Pro Pro Gly Pro Lys Arg Gly
465 470 475 480
Gly Phe Glu Ser Tyr Gln Glu Arg Val Glu Gly Asn Lys Val Arg Glu
485 490 495
Arg Ser Pro Ser Phe Gly Glu Tyr Tyr Ser His Pro Arg Leu Phe Trp
500 505 510
Leu Ser Gln Thr Pro Phe Glu Gln Arg His Ile Val Asp Gly Phe Ser
515 520 525
Phe Glu Leu Ser Lys Val Val Arg Pro Tyr Ile Arg Glu Arg Val Val
530 535 540
Asp Gln Leu Ala His Ile Asp Leu Thr Leu Ala Gln Ala Val Ala Lys
545 550 555 560
Asn Leu Gly Ile Glu Leu Thr Asp Asp Gln Leu Asn Ile Thr Pro Pro
565 570 575
Pro Asp Val Asn Gly Leu Lys Lys Asp Pro Ser Leu Ser Leu Tyr Ala
580 585 590
Ile Pro Asp Gly Asp Val Lys Gly Arg Val Val Ala Ile Leu Leu Asn
595 600 605
Asp Glu Val Arg Ser Ala Asp Leu Leu Ala Ile Leu Lys Ala Leu Lys
610 615 620
Ala Lys Gly Val His Ala Lys Leu Leu Tyr Ser Arg Met Gly Glu Val
625 630 635 640
Thr Ala Asp Asp Gly Thr Val Leu Pro Ile Ala Ala Thr Phe Ala Gly
645 650 655
Ala Pro Ser Leu Thr Val Asp Ala Val Ile Val Pro Cys Gly Asn Ile
660 665 670
Ala Asp Ile Ala Asp Asn Gly Asp Ala Asn Tyr Tyr Leu Met Glu Ala
675 680 685
Tyr Lys His Leu Lys Pro Ile Ala Leu Ala Gly Asp Ala Arg Lys Phe
690 695 700
Lys Ala Thr Ile Lys Ile Ala Asp Gln Gly Glu Glu Gly Ile Val Glu
705 710 715 720
Ala Asp Ser Ala Asp Gly Ser Phe Met Asp Glu Leu Leu Thr Leu Met
725 730 735
Ala Ala His Arg Val Trp Ser Arg Ile Pro Lys Ile Asp Lys Ile Pro
740 745 750
Ala
<210> 4
<211> 108
<212> PRT
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 4
Met Asn Ile Ser Ser Leu Arg Lys Ala Phe Ile Phe Met Gly Ala Val
1 5 10 15
Ala Ala Leu Ser Leu Val Asn Ala Gln Ser Ala Leu Ala Ala Asn Glu
20 25 30
Ser Ala Lys Asp Met Thr Cys Gln Glu Phe Ile Asp Leu Asn Pro Lys
35 40 45
Ala Met Thr Pro Val Ala Trp Trp Met Leu His Glu Glu Thr Val Tyr
50 55 60
Lys Gly Gly Asp Thr Val Thr Leu Asn Glu Thr Asp Leu Thr Gln Ile
65 70 75 80
Pro Lys Val Ile Glu Tyr Cys Lys Lys Asn Pro Gln Lys Asn Leu Tyr
85 90 95
Thr Phe Lys Asn Gln Ala Ser Asn Asp Leu Pro Asn
100 105
<210> 5
<211> 528
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 5
atgatttttc tcatgacgaa agattctttt cttttacagg gcttttggca gttgaaagat 60
aatcacgaaa tgataaaaat caattccctg tcagagatca aaaaagtagg caataaaccc 120
ttcaaggtta tcattgatac ctatcacaat catatccttg atgaagaagc gattaaattt 180
ctggagaaat tagatgccga gagaattatt gttttggcac cttatcacat cagtaaacta 240
aaagctaaag cgcctattta ttttgttagc cgcaaagaaa gtatcaaaaa tcttcttgag 300
attacttatg gtaaacactt gccccataag aattcacaat tatgtttttc acataatcag 360
ttcaaaatta tgcaactgat tctgaaaaat aaaaatgaaa gcaatatcac gtcgacgctc 420
aatatttcgc aacaaacatt aaagattcag aaattcaaca ttatgtacaa gctgaaacta 480
agacgtatga gcgacatcgt caccctgggt atcacatctt atttttag 528
<210> 6
<211> 582
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 6
atgtcattcg aattacctgc actaccatat gctaaagatg ctctggcacc gcacatttct 60
gcggaaacca tcgagtatca ctacggcaag caccatcaga cttatgtcac taacctgaac 120
aacctgatta aaggtaccgc gtttgaaggt aaatcactgg aagagattat tcgcagctct 180
gaaggtggcg tattcaacaa cgcagctcag gtctggaacc atactttcta ctggaactgc 240
ctggcaccga acgccggtgg cgaaccgact ggaaaagtcg ctgaagctat cgccgcatct 300
tttggcagct ttgccgattt caaagcgcag tttactgatg cagcgatcaa aaactttggt 360
tctggctgga cctggctggt gaaaaacagc gatggcaaac tggctatcgt ttcaacctct 420
aacgcgggta ctccgctgac caccgatgcg actccgctgc tgaccgttga tgtctgggaa 480
cacgcttatt acatcgacta tcgcaatgca cgtcctggct atctggagca cttctgggcg 540
ctggtgaact gggaattcgt agcgaaaaat ctcgctgcat aa 582
<210> 7
<211> 2262
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 7
atgtcgcaac ataacgaaaa gaacccacat cagcaccagt caccactaca cgattccagc 60
gaagcgaaac cggggatgga ctcactggca cctgaggacg gctctcatcg tccagcggct 120
gaaccaacac cgccaggtgc acaacctacc gccccaggga gcctgaaagc ccctgatacg 180
cgtaacgaaa aacttaattc tctggaagac gtacgcaaag gcagtgaaaa ttatgcgctg 240
accactaatc agggcgtgcg catcgccgac gatcaaaact cactgcgtgc cggtagccgt 300
ggtccaacgc tgctggaaga ttttattctg cgcgagaaaa tcacccactt tgaccatgag 360
cgcattccgg aacgtattgt tcatgcacgc ggatcagccg ctcacggtta tttccagcca 420
tataaaagct taagcgatat taccaaagcg gatttcctct cagatccgaa caaaatcacc 480
ccagtatttg tacgtttctc taccgttcag ggtggtgctg gctctgctga taccgtgcgt 540
gatatccgtg gctttgccac caagttctat accgaagagg gtatttttga cctcgttggc 600
aataacacgc caatcttctt tatccaggat gcgcataaat tccccgattt tgttcatgcg 660
gtaaaaccag aaccgcactg ggcaattcca caagggcaaa gtgcccacga tactttctgg 720
gattatgttt ctctgcaacc tgaaactctg cacaacgtga tgtgggcgat gtcggatcgc 780
ggcatccccc gcagttaccg caccatggaa ggcttcggta ttcacacctt ccgcctgatt 840
aatgccgaag ggaaggcaac gtttgtacgt ttccactgga aaccactggc aggtaaagcc 900
tcactcgttt gggatgaagc acaaaaactc accggacgtg acccggactt ccaccgccgc 960
gagttgtggg aagccattga agcaggcgat tttccggaat acgaactggg cttccagttg 1020
attcctgaag aagatgaatt caagttcgac ttcgatcttc tcgatccaac caaacttatc 1080
ccggaagaac tggtgcccgt tcagcgtgtc ggcaaaatgg tgctcaatcg caacccggat 1140
aacttctttg ctgaaaacga acaggcggct ttccatcctg ggcatatcgt gccgggactg 1200
gacttcacca acgatccgct gttgcaggga cgtttgttct cctataccga tacacaaatc 1260
agtcgtcttg gtgggccgaa tttccatgag attccgatta accgtccgac ctgcccttac 1320
cataatttcc agcgtgacgg catgcatcgc atggggatcg acactaaccc ggcgaattac 1380
gaaccgaact cgattaacga taactggccg cgcgaaacac cgccggggcc gaaacgcggc 1440
ggttttgaat cataccagga gcgcgtggaa ggcaataaag ttcgcgagcg cagcccatcg 1500
tttggcgaat attattccca tccgcgtctg ttctggctaa gtcagacgcc atttgagcag 1560
cgccatattg tcgatggttt cagttttgag ttaagcaaag tcgttcgtcc gtatattcgt 1620
gagcgcgttg ttgaccagct ggcgcatatt gatctcactc tggcccaggc ggtggcgaaa 1680
aatctcggta tcgaactgac tgacgaccag ctgaatatca ccccacctcc ggacgtcaac 1740
ggtctgaaaa aggatccatc cttaagtttg tacgccattc ctgacggtga tgtgaaaggt 1800
cgcgtggtag cgattttact taatgatgaa gtgagatcgg cagaccttct ggccattctc 1860
aaggcgctga aggccaaagg cgttcatgcc aaactgctct actcccgaat gggtgaagtg 1920
actgcggatg acggtacggt gttgcctata gccgctacct ttgccggtgc accttcgctg 1980
acggtcgatg cggtcattgt cccttgcggc aatatcgcgg atatcgctga caacggcgat 2040
gccaactact acctgatgga agcctacaaa caccttaaac cgattgcgct ggcgggtgac 2100
gcgcgcaagt ttaaagcaac aatcaagatc gctgaccagg gtgaagaagg gattgtggaa 2160
gctgacagcg ctgacggtag ttttatggat gaactgctaa cgctgatggc agcacaccgc 2220
gtgtggtcac gcattcctaa gattgacaaa attcctgcct ga 2262
<210> 8
<211> 327
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 8
atgaatattt catctctccg taaagcgttt atttttatgg gcgctgtagc ggctttgtca 60
ctggtgaacg cacaatctgc gttggcagcc aatgaatccg ctaaagatat gacctgccag 120
gaatttattg atctgaatcc aaaagcaatg accccggttg catggtggat gctgcatgaa 180
gaaacagtat ataaaggtgg cgataccgtt actttaaatg aaaccgatct cactcaaatt 240
cctaaagtga tcgaatactg taagaaaaac ccgcagaaaa atttgtatac cttcaaaaat 300
caagcatcta atgacttgcc gaattaa 327
<210> 9
<211> 247
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 9
aggcatcaaa taaaacgaaa ggctcagtcg gaagactggg cctttcgttt tatctgttgt 60
ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg 120
aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt 180
aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tcctgtcgtc 240
atatcta 247
<210> 10
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 10
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
cccagggata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 11
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 11
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 12
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 12
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 13
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 13
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 14
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 14
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
agccacgcta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 15
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 15
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
gtattaatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 16
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 16
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
aagccgtcta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 17
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 17
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
aactaaatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 18
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 18
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
attatacgta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 19
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 19
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gacgcatcta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 20
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 20
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
agccgctgta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 21
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 21
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
tgaacggata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 22
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 22
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
ttacacacta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 23
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 23
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
cttgtcggta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 24
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 24
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
atcaagaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 25
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 25
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
tcaacagata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 26
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 26
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
cagctcgata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 27
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 27
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
gtattaatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 28
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 28
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
ccaatgatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 29
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 29
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
agaaaaggta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 30
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 30
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
aagcatatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 31
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 31
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
accaataata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 32
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 32
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acccaaacta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 33
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 33
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
ttagacaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 34
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 34
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
cgtaagaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 35
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 35
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
ctaacaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 36
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 36
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
aaaaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 37
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 37
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
tagaatttta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 38
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 38
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
agcactaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 39
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 39
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
tagactcata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 40
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 40
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgatagata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 41
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 41
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
gtaataaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 42
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 42
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
ggggttcgta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 43
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 43
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gtggaaccta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 44
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 44
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
gaaaattata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 45
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 45
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
atgacaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 46
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 46
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
tgtaaattta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 47
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 47
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
gcatatcata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 48
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 48
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctaacctata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 49
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 49
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
gttgaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 50
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 50
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
ctaaaattta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 51
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 51
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaatta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 52
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 52
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acaaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 53
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 53
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
gggaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 54
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 54
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
gtggtcgata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 55
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 55
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
atgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 56
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 56
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatt 60
aggaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 57
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 57
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ttgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 58
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 58
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
tccaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 59
<211> 129
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 59
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctcagaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgaca 129
<210> 60
<211> 886
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 60
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aaacaaaatg aggaggtact gagcatatga tttttctcat 120
gacgaaagat tcttttcttt tacagggctt ttggcagttg aaagataatc acgaaatgat 180
aaaaatcaat tccctgtcag agatcaaaaa agtaggcaat aaacccttca aggttatcat 240
tgatacctat cacaatcata tccttgatga agaagcgatt aaatttctgg agaaattaga 300
tgccgagaga attattgttt tggcacctta tcacatcagt aaactaaaag ctaaagcgcc 360
tatttatttt gttagccgca aagaaagtat caaaaatctt cttgagatta cttatggtaa 420
acacttgccc cataagaatt cacaattatg tttttcacat aatcagttca aaattatgca 480
actgattctg aaaaataaaa atgaaagcaa tatcacgtcg acgctcaata tttcgcaaca 540
aacattaaag attcagaaat tcaacattat gtacaagctg aaactaagac gtatgagcga 600
catcgtcacc ctgggtatca catcttattt ttagctcgag aggcatcaaa taaaacgaaa 660
ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctccg 720
agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg 780
cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg 840
gatggccttt ttgcgtttct acaaactctt cctgtcgtca tatcta 886
<210> 61
<211> 886
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 61
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aaacaaaatg aggaggtact gagcatatga tttttctcat 120
gacgaaagat tcttttcttt tacagggctt ttggcagttg aaagataatc acgaaatgat 180
aaaaatcaat tccctgtcag agatcaaaaa agtaggcaat aaacccttca aggttatcat 240
tgatacctat cacaatcata tccttgatga agaagcgatt aaatttctgg agaaattaga 300
tgccgagaga attattgttt tggcacctta tcacatcagt aaactaaaag ctaaagcgcc 360
tatttatttt gttagccgca aagaaagtat caaaaatctt cttgagatta cttatggtaa 420
acacttgccc cataagaatt cacaattatg tttttcacat aatcagttca aaattatgca 480
actgattctg aaaaataaaa atgaaagcaa tatcacgtcg acgctcaata tttcgcaaca 540
aacattaaag attcagaaat tcaacattat gtacaagctg aaactaagac gtatgagcga 600
catcgtcacc ctgggtatca catcttattt ttagctcgag aggcatcaaa taaaacgaaa 660
ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctccg 720
agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg 780
cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg 840
gatggccttt ttgcgtttct acaaactctt cctgtcgtca tatcta 886
<210> 62
<211> 886
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 62
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aaacaaaatg aggaggtact gagcatatga tttttctcat 120
gacgaaagat tcttttcttt tacagggctt ttggcagttg aaagataatc acgaaatgat 180
aaaaatcaat tccctgtcag agatcaaaaa agtaggcaat aaacccttca aggttatcat 240
tgatacctat cacaatcata tccttgatga agaagcgatt aaatttctgg agaaattaga 300
tgccgagaga attattgttt tggcacctta tcacatcagt aaactaaaag ctaaagcgcc 360
tatttatttt gttagccgca aagaaagtat caaaaatctt cttgagatta cttatggtaa 420
acacttgccc cataagaatt cacaattatg tttttcacat aatcagttca aaattatgca 480
actgattctg aaaaataaaa atgaaagcaa tatcacgtcg acgctcaata tttcgcaaca 540
aacattaaag attcagaaat tcaacattat gtacaagctg aaactaagac gtatgagcga 600
catcgtcacc ctgggtatca catcttattt ttagctcgag aggcatcaaa taaaacgaaa 660
ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctccg 720
agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg 780
cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg 840
gatggccttt ttgcgtttct acaaactctt cctgtcgtca tatcta 886
<210> 63
<211> 963
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 63
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcattcga attacctgca ctaccatatg ctaaagatgc tctggcaccg 180
cacatttctg cggaaaccat cgagtatcac tacggcaagc accatcagac ttatgtcact 240
aacctgaaca acctgattaa aggtaccgcg tttgaaggta aatcactgga agagattatt 300
cgcagctctg aaggtggcgt attcaacaac gcagctcagg tctggaacca tactttctac 360
tggaactgcc tggcaccgaa cgccggtggc gaaccgactg gaaaagtcgc tgaagctatc 420
gccgcatctt ttggcagctt tgccgatttc aaagcgcagt ttactgatgc agcgatcaaa 480
aactttggtt ctggctggac ctggctggtg aaaaacagcg atggcaaact ggctatcgtt 540
tcaacctcta acgcgggtac tccgctgacc accgatgcga ctccgctgct gaccgttgat 600
gtctgggaac acgcttatta catcgactat cgcaatgcac gtcctggcta tctggagcac 660
ttctgggcgc tggtgaactg ggaattcgta gcgaaaaatc tcgctgcata actcgagagg 720
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 780
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 840
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 900
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 960
cta 963
<210> 64
<211> 963
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 64
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcattcga attacctgca ctaccatatg ctaaagatgc tctggcaccg 180
cacatttctg cggaaaccat cgagtatcac tacggcaagc accatcagac ttatgtcact 240
aacctgaaca acctgattaa aggtaccgcg tttgaaggta aatcactgga agagattatt 300
cgcagctctg aaggtggcgt attcaacaac gcagctcagg tctggaacca tactttctac 360
tggaactgcc tggcaccgaa cgccggtggc gaaccgactg gaaaagtcgc tgaagctatc 420
gccgcatctt ttggcagctt tgccgatttc aaagcgcagt ttactgatgc agcgatcaaa 480
aactttggtt ctggctggac ctggctggtg aaaaacagcg atggcaaact ggctatcgtt 540
tcaacctcta acgcgggtac tccgctgacc accgatgcga ctccgctgct gaccgttgat 600
gtctgggaac acgcttatta catcgactat cgcaatgcac gtcctggcta tctggagcac 660
ttctgggcgc tggtgaactg ggaattcgta gcgaaaaatc tcgctgcata actcgagagg 720
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 780
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 840
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 900
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 960
cta 963
<210> 65
<211> 963
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 65
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcattcga attacctgca ctaccatatg ctaaagatgc tctggcaccg 180
cacatttctg cggaaaccat cgagtatcac tacggcaagc accatcagac ttatgtcact 240
aacctgaaca acctgattaa aggtaccgcg tttgaaggta aatcactgga agagattatt 300
cgcagctctg aaggtggcgt attcaacaac gcagctcagg tctggaacca tactttctac 360
tggaactgcc tggcaccgaa cgccggtggc gaaccgactg gaaaagtcgc tgaagctatc 420
gccgcatctt ttggcagctt tgccgatttc aaagcgcagt ttactgatgc agcgatcaaa 480
aactttggtt ctggctggac ctggctggtg aaaaacagcg atggcaaact ggctatcgtt 540
tcaacctcta acgcgggtac tccgctgacc accgatgcga ctccgctgct gaccgttgat 600
gtctgggaac acgcttatta catcgactat cgcaatgcac gtcctggcta tctggagcac 660
ttctgggcgc tggtgaactg ggaattcgta gcgaaaaatc tcgctgcata actcgagagg 720
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 780
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 840
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 900
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 960
cta 963
<210> 66
<211> 2643
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 66
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg actcgagagg 2400
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 2460
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 2520
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 2580
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 2640
cta 2643
<210> 67
<211> 2643
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 67
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg actcgagagg 2400
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 2460
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 2520
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 2580
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 2640
cta 2643
<210> 68
<211> 2643
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 68
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg actcgagagg 2400
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 2460
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 2520
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 2580
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 2640
cta 2643
<210> 69
<211> 685
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 69
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aaacaaaatg aggaggtact gagcatatga atatttcatc 120
tctccgtaaa gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca 180
atctgcgttg gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct 240
gaatccaaaa gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa 300
aggtggcgat accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga 360
atactgtaag aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga 420
cttgccgaat taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 480
ctttcgtttt atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga 540
gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa 600
actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta 660
caaactcttc ctgtcgtcat atcta 685
<210> 70
<211> 685
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 70
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aaacaaaatg aggaggtact gagcatatga atatttcatc 120
tctccgtaaa gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca 180
atctgcgttg gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct 240
gaatccaaaa gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa 300
aggtggcgat accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga 360
atactgtaag aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga 420
cttgccgaat taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 480
ctttcgtttt atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga 540
gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa 600
actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta 660
caaactcttc ctgtcgtcat atcta 685
<210> 71
<211> 685
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 71
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aaacaaaatg aggaggtact gagcatatga atatttcatc 120
tctccgtaaa gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca 180
atctgcgttg gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct 240
gaatccaaaa gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa 300
aggtggcgat accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga 360
atactgtaag aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga 420
cttgccgaat taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 480
ctttcgtttt atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga 540
gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa 600
actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta 660
caaactcttc ctgtcgtcat atcta 685
<210> 72
<211> 3298
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 72
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg acgcaggtgt 2400
ggttatgcta ctagagacgg ccccgcagtg gggttaaatg aaaaaacaaa ttgagggtat 2460
gacaatgtca ttcgaattac ctgcactacc atatgctaaa gatgctctgg caccgcacat 2520
ttctgcggaa accatcgagt atcactacgg caagcaccat cagacttatg tcactaacct 2580
gaacaacctg attaaaggta ccgcgtttga aggtaaatca ctggaagaga ttattcgcag 2640
ctctgaaggt ggcgtattca acaacgcagc tcaggtctgg aaccatactt tctactggaa 2700
ctgcctggca ccgaacgccg gtggcgaacc gactggaaaa gtcgctgaag ctatcgccgc 2760
atcttttggc agctttgccg atttcaaagc gcagtttact gatgcagcga tcaaaaactt 2820
tggttctggc tggacctggc tggtgaaaaa cagcgatggc aaactggcta tcgtttcaac 2880
ctctaacgcg ggtactccgc tgaccaccga tgcgactccg ctgctgaccg ttgatgtctg 2940
ggaacacgct tattacatcg actatcgcaa tgcacgtcct ggctatctgg agcacttctg 3000
ggcgctggtg aactgggaat tcgtagcgaa aaatctcgct gcataactcg agaggcatca 3060
aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt 3120
gaacgctctc cgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 3180
cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 3240
gccatcctga cggatggcct ttttgcgttt ctacaaactc ttcctgtcgt catatcta 3298
<210> 73
<211> 3298
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 73
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg acgcaggtgt 2400
ggttatgcta ctagagacgg ccccgcagtg gggttaaatg aaaaaacaaa ttgagggtat 2460
gacaatgtca ttcgaattac ctgcactacc atatgctaaa gatgctctgg caccgcacat 2520
ttctgcggaa accatcgagt atcactacgg caagcaccat cagacttatg tcactaacct 2580
gaacaacctg attaaaggta ccgcgtttga aggtaaatca ctggaagaga ttattcgcag 2640
ctctgaaggt ggcgtattca acaacgcagc tcaggtctgg aaccatactt tctactggaa 2700
ctgcctggca ccgaacgccg gtggcgaacc gactggaaaa gtcgctgaag ctatcgccgc 2760
atcttttggc agctttgccg atttcaaagc gcagtttact gatgcagcga tcaaaaactt 2820
tggttctggc tggacctggc tggtgaaaaa cagcgatggc aaactggcta tcgtttcaac 2880
ctctaacgcg ggtactccgc tgaccaccga tgcgactccg ctgctgaccg ttgatgtctg 2940
ggaacacgct tattacatcg actatcgcaa tgcacgtcct ggctatctgg agcacttctg 3000
ggcgctggtg aactgggaat tcgtagcgaa aaatctcgct gcataactcg agaggcatca 3060
aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt 3120
gaacgctctc cgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 3180
cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 3240
gccatcctga cggatggcct ttttgcgttt ctacaaactc ttcctgtcgt catatcta 3298
<210> 74
<211> 3298
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 74
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
ctgaaaaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc agcaccagtc accactacac 180
gattccagcg aagcgaaacc ggggatggac tcactggcac ctgaggacgg ctctcatcgt 240
ccagcggctg aaccaacacc gccaggtgca caacctaccg ccccagggag cctgaaagcc 300
cctgatacgc gtaacgaaaa acttaattct ctggaagacg tacgcaaagg cagtgaaaat 360
tatgcgctga ccactaatca gggcgtgcgc atcgccgacg atcaaaactc actgcgtgcc 420
ggtagccgtg gtccaacgct gctggaagat tttattctgc gcgagaaaat cacccacttt 480
gaccatgagc gcattccgga acgtattgtt catgcacgcg gatcagccgc tcacggttat 540
ttccagccat ataaaagctt aagcgatatt accaaagcgg atttcctctc agatccgaac 600
aaaatcaccc cagtatttgt acgtttctct accgttcagg gtggtgctgg ctctgctgat 660
accgtgcgtg atatccgtgg ctttgccacc aagttctata ccgaagaggg tatttttgac 720
ctcgttggca ataacacgcc aatcttcttt atccaggatg cgcataaatt ccccgatttt 780
gttcatgcgg taaaaccaga accgcactgg gcaattccac aagggcaaag tgcccacgat 840
actttctggg attatgtttc tctgcaacct gaaactctgc acaacgtgat gtgggcgatg 900
tcggatcgcg gcatcccccg cagttaccgc accatggaag gcttcggtat tcacaccttc 960
cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt tccactggaa accactggca 1020
ggtaaagcct cactcgtttg ggatgaagca caaaaactca ccggacgtga cccggacttc 1080
caccgccgcg agttgtggga agccattgaa gcaggcgatt ttccggaata cgaactgggc 1140
ttccagttga ttcctgaaga agatgaattc aagttcgact tcgatcttct cgatccaacc 1200
aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg gcaaaatggt gctcaatcgc 1260
aacccggata acttctttgc tgaaaacgaa caggcggctt tccatcctgg gcatatcgtg 1320
ccgggactgg acttcaccaa cgatccgctg ttgcagggac gtttgttctc ctataccgat 1380
acacaaatca gtcgtcttgg tgggccgaat ttccatgaga ttccgattaa ccgtccgacc 1440
tgcccttacc ataatttcca gcgtgacggc atgcatcgca tggggatcga cactaacccg 1500
gcgaattacg aaccgaactc gattaacgat aactggccgc gcgaaacacc gccggggccg 1560
aaacgcggcg gttttgaatc ataccaggag cgcgtggaag gcaataaagt tcgcgagcgc 1620
agcccatcgt ttggcgaata ttattcccat ccgcgtctgt tctggctaag tcagacgcca 1680
tttgagcagc gccatattgt cgatggtttc agttttgagt taagcaaagt cgttcgtccg 1740
tatattcgtg agcgcgttgt tgaccagctg gcgcatattg atctcactct ggcccaggcg 1800
gtggcgaaaa atctcggtat cgaactgact gacgaccagc tgaatatcac cccacctccg 1860
gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt acgccattcc tgacggtgat 1920
gtgaaaggtc gcgtggtagc gattttactt aatgatgaag tgagatcggc agaccttctg 1980
gccattctca aggcgctgaa ggccaaaggc gttcatgcca aactgctcta ctcccgaatg 2040
ggtgaagtga ctgcggatga cggtacggtg ttgcctatag ccgctacctt tgccggtgca 2100
ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca atatcgcgga tatcgctgac 2160
aacggcgatg ccaactacta cctgatggaa gcctacaaac accttaaacc gattgcgctg 2220
gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg ctgaccaggg tgaagaaggg 2280
attgtggaag ctgacagcgc tgacggtagt tttatggatg aactgctaac gctgatggca 2340
gcacaccgcg tgtggtcacg cattcctaag attgacaaaa ttcctgcctg acgcaggtgt 2400
ggttatgcta ctagagacgg ccccgcagtg gggttaaatg aaaaaacaaa ttgagggtat 2460
gacaatgtca ttcgaattac ctgcactacc atatgctaaa gatgctctgg caccgcacat 2520
ttctgcggaa accatcgagt atcactacgg caagcaccat cagacttatg tcactaacct 2580
gaacaacctg attaaaggta ccgcgtttga aggtaaatca ctggaagaga ttattcgcag 2640
ctctgaaggt ggcgtattca acaacgcagc tcaggtctgg aaccatactt tctactggaa 2700
ctgcctggca ccgaacgccg gtggcgaacc gactggaaaa gtcgctgaag ctatcgccgc 2760
atcttttggc agctttgccg atttcaaagc gcagtttact gatgcagcga tcaaaaactt 2820
tggttctggc tggacctggc tggtgaaaaa cagcgatggc aaactggcta tcgtttcaac 2880
ctctaacgcg ggtactccgc tgaccaccga tgcgactccg ctgctgaccg ttgatgtctg 2940
ggaacacgct tattacatcg actatcgcaa tgcacgtcct ggctatctgg agcacttctg 3000
ggcgctggtg aactgggaat tcgtagcgaa aaatctcgct gcataactcg agaggcatca 3060
aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt 3120
gaacgctctc cgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 3180
cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 3240
gccatcctga cggatggcct ttttgcgttt ctacaaactc ttcctgtcgt catatcta 3298
<210> 75
<211> 4578
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 75
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcattcga attacctgca ctaccatatg ctaaagatgc tctggcaccg 180
cacatttctg cggaaaccat cgagtatcac tacggcaagc accatcagac ttatgtcact 240
aacctgaaca acctgattaa aggtaccgcg tttgaaggta aatcactgga agagattatt 300
cgcagctctg aaggtggcgt attcaacaac gcagctcagg tctggaacca tactttctac 360
tggaactgcc tggcaccgaa cgccggtggc gaaccgactg gaaaagtcgc tgaagctatc 420
gccgcatctt ttggcagctt tgccgatttc aaagcgcagt ttactgatgc agcgatcaaa 480
aactttggtt ctggctggac ctggctggtg aaaaacagcg atggcaaact ggctatcgtt 540
tcaacctcta acgcgggtac tccgctgacc accgatgcga ctccgctgct gaccgttgat 600
gtctgggaac acgcttatta catcgactat cgcaatgcac gtcctggcta tctggagcac 660
ttctgggcgc tggtgaactg ggaattcgta gcgaaaaatc tcgctgcata actcgagagg 720
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 780
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 840
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 900
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 960
ctaatctatt cagcgtttgt acatatcgtt acacgctgaa accaaccact cacggaagtc 1020
tgccatactg aaaaatatag ttatttcaac ggccccgcag tggggttaaa tgaaaaaaca 1080
aattgagggt atgacaatgt cgcaacataa cgaaaagaac ccacatcagc accagtcacc 1140
actacacgat tccagcgaag cgaaaccggg gatggactca ctggcacctg aggacggctc 1200
tcatcgtcca gcggctgaac caacaccgcc aggtgcacaa cctaccgccc cagggagcct 1260
gaaagcccct gatacgcgta acgaaaaact taattctctg gaagacgtac gcaaaggcag 1320
tgaaaattat gcgctgacca ctaatcaggg cgtgcgcatc gccgacgatc aaaactcact 1380
gcgtgccggt agccgtggtc caacgctgct ggaagatttt attctgcgcg agaaaatcac 1440
ccactttgac catgagcgca ttccggaacg tattgttcat gcacgcggat cagccgctca 1500
cggttatttc cagccatata aaagcttaag cgatattacc aaagcggatt tcctctcaga 1560
tccgaacaaa atcaccccag tatttgtacg tttctctacc gttcagggtg gtgctggctc 1620
tgctgatacc gtgcgtgata tccgtggctt tgccaccaag ttctataccg aagagggtat 1680
ttttgacctc gttggcaata acacgccaat cttctttatc caggatgcgc ataaattccc 1740
cgattttgtt catgcggtaa aaccagaacc gcactgggca attccacaag ggcaaagtgc 1800
ccacgatact ttctgggatt atgtttctct gcaacctgaa actctgcaca acgtgatgtg 1860
ggcgatgtcg gatcgcggca tcccccgcag ttaccgcacc atggaaggct tcggtattca 1920
caccttccgc ctgattaatg ccgaagggaa ggcaacgttt gtacgtttcc actggaaacc 1980
actggcaggt aaagcctcac tcgtttggga tgaagcacaa aaactcaccg gacgtgaccc 2040
ggacttccac cgccgcgagt tgtgggaagc cattgaagca ggcgattttc cggaatacga 2100
actgggcttc cagttgattc ctgaagaaga tgaattcaag ttcgacttcg atcttctcga 2160
tccaaccaaa cttatcccgg aagaactggt gcccgttcag cgtgtcggca aaatggtgct 2220
caatcgcaac ccggataact tctttgctga aaacgaacag gcggctttcc atcctgggca 2280
tatcgtgccg ggactggact tcaccaacga tccgctgttg cagggacgtt tgttctccta 2340
taccgataca caaatcagtc gtcttggtgg gccgaatttc catgagattc cgattaaccg 2400
tccgacctgc ccttaccata atttccagcg tgacggcatg catcgcatgg ggatcgacac 2460
taacccggcg aattacgaac cgaactcgat taacgataac tggccgcgcg aaacaccgcc 2520
ggggccgaaa cgcggcggtt ttgaatcata ccaggagcgc gtggaaggca ataaagttcg 2580
cgagcgcagc ccatcgtttg gcgaatatta ttcccatccg cgtctgttct ggctaagtca 2640
gacgccattt gagcagcgcc atattgtcga tggtttcagt tttgagttaa gcaaagtcgt 2700
tcgtccgtat attcgtgagc gcgttgttga ccagctggcg catattgatc tcactctggc 2760
ccaggcggtg gcgaaaaatc tcggtatcga actgactgac gaccagctga atatcacccc 2820
acctccggac gtcaacggtc tgaaaaagga tccatcctta agtttgtacg ccattcctga 2880
cggtgatgtg aaaggtcgcg tggtagcgat tttacttaat gatgaagtga gatcggcaga 2940
ccttctggcc attctcaagg cgctgaaggc caaaggcgtt catgccaaac tgctctactc 3000
ccgaatgggt gaagtgactg cggatgacgg tacggtgttg cctatagccg ctacctttgc 3060
cggtgcacct tcgctgacgg tcgatgcggt cattgtccct tgcggcaata tcgcggatat 3120
cgctgacaac ggcgatgcca actactacct gatggaagcc tacaaacacc ttaaaccgat 3180
tgcgctggcg ggtgacgcgc gcaagtttaa agcaacaatc aagatcgctg accagggtga 3240
agaagggatt gtggaagctg acagcgctga cggtagtttt atggatgaac tgctaacgct 3300
gatggcagca caccgcgtgt ggtcacgcat tcctaagatt gacaaaattc ctgcctgact 3360
cgagaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg 3420
ttgtttgtcg gtgaacgctc tccgagtagg acaaatccgc cgggagcgga tttgaacgtt 3480
gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa 3540
attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttcctgtc 3600
gtcatatcta gcttattcag cgtttgtaca tatcgttaca cgctgaaacc aaccactcac 3660
ggaagtctgc catactgaaa aatatagtta tttcaacggc cccgcagtgg ggttaaatga 3720
aaaaacaaat tgagggtatg acaatgaata tttcatctct ccgtaaagcg tttattttta 3780
tgggcgctgt agcggctttg tcactggtga acgcacaatc tgcgttggca gccaatgaat 3840
ccgctaaaga tatgacctgc caggaattta ttgatctgaa tccaaaagca atgaccccgg 3900
ttgcatggtg gatgctgcat gaagaaacag tatataaagg tggcgatacc gttactttaa 3960
atgaaaccga tctcactcaa attcctaaag tgatcgaata ctgtaagaaa aacccgcaga 4020
aaaatttgta taccttcaaa aatcaagcat ctaatgactt gccgaattaa ctcgagaggc 4080
atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 4140
cggtgaacgc tctccgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 4200
acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca 4260
gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg tcgtcatatc 4320
tagagtctcg agaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 4380
tttatctgtt gtttgtcggt gaacgctctc cgagtaggac aaatccgccg ggagcggatt 4440
tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 4500
ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 4560
ttcctgtcgt catatcta 4578
<210> 76
<211> 4578
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 76
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgtcattcga attacctgca ctaccatatg ctaaagatgc tctggcaccg 180
cacatttctg cggaaaccat cgagtatcac tacggcaagc accatcagac ttatgtcact 240
aacctgaaca acctgattaa aggtaccgcg tttgaaggta aatcactgga agagattatt 300
cgcagctctg aaggtggcgt attcaacaac gcagctcagg tctggaacca tactttctac 360
tggaactgcc tggcaccgaa cgccggtggc gaaccgactg gaaaagtcgc tgaagctatc 420
gccgcatctt ttggcagctt tgccgatttc aaagcgcagt ttactgatgc agcgatcaaa 480
aactttggtt ctggctggac ctggctggtg aaaaacagcg atggcaaact ggctatcgtt 540
tcaacctcta acgcgggtac tccgctgacc accgatgcga ctccgctgct gaccgttgat 600
gtctgggaac acgcttatta catcgactat cgcaatgcac gtcctggcta tctggagcac 660
ttctgggcgc tggtgaactg ggaattcgta gcgaaaaatc tcgctgcata actcgagagg 720
catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 780
tcggtgaacg ctctccgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 840
aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 900
agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcct gtcgtcatat 960
ctaatctatt cagcgtttgt acatatcgtt acacgctgaa accaaccact cacggaagtc 1020
tgccatactg aaaaatatag ttatttcaac ggccccgcag tggggttaaa tgaaaaaaca 1080
aattgagggt atgacaatgt cgcaacataa cgaaaagaac ccacatcagc accagtcacc 1140
actacacgat tccagcgaag cgaaaccggg gatggactca ctggcacctg aggacggctc 1200
tcatcgtcca gcggctgaac caacaccgcc aggtgcacaa cctaccgccc cagggagcct 1260
gaaagcccct gatacgcgta acgaaaaact taattctctg gaagacgtac gcaaaggcag 1320
tgaaaattat gcgctgacca ctaatcaggg cgtgcgcatc gccgacgatc aaaactcact 1380
gcgtgccggt agccgtggtc caacgctgct ggaagatttt attctgcgcg agaaaatcac 1440
ccactttgac catgagcgca ttccggaacg tattgttcat gcacgcggat cagccgctca 1500
cggttatttc cagccatata aaagcttaag cgatattacc aaagcggatt tcctctcaga 1560
tccgaacaaa atcaccccag tatttgtacg tttctctacc gttcagggtg gtgctggctc 1620
tgctgatacc gtgcgtgata tccgtggctt tgccaccaag ttctataccg aagagggtat 1680
ttttgacctc gttggcaata acacgccaat cttctttatc caggatgcgc ataaattccc 1740
cgattttgtt catgcggtaa aaccagaacc gcactgggca attccacaag ggcaaagtgc 1800
ccacgatact ttctgggatt atgtttctct gcaacctgaa actctgcaca acgtgatgtg 1860
ggcgatgtcg gatcgcggca tcccccgcag ttaccgcacc atggaaggct tcggtattca 1920
caccttccgc ctgattaatg ccgaagggaa ggcaacgttt gtacgtttcc actggaaacc 1980
actggcaggt aaagcctcac tcgtttggga tgaagcacaa aaactcaccg gacgtgaccc 2040
ggacttccac cgccgcgagt tgtgggaagc cattgaagca ggcgattttc cggaatacga 2100
actgggcttc cagttgattc ctgaagaaga tgaattcaag ttcgacttcg atcttctcga 2160
tccaaccaaa cttatcccgg aagaactggt gcccgttcag cgtgtcggca aaatggtgct 2220
caatcgcaac ccggataact tctttgctga aaacgaacag gcggctttcc atcctgggca 2280
tatcgtgccg ggactggact tcaccaacga tccgctgttg cagggacgtt tgttctccta 2340
taccgataca caaatcagtc gtcttggtgg gccgaatttc catgagattc cgattaaccg 2400
tccgacctgc ccttaccata atttccagcg tgacggcatg catcgcatgg ggatcgacac 2460
taacccggcg aattacgaac cgaactcgat taacgataac tggccgcgcg aaacaccgcc 2520
ggggccgaaa cgcggcggtt ttgaatcata ccaggagcgc gtggaaggca ataaagttcg 2580
cgagcgcagc ccatcgtttg gcgaatatta ttcccatccg cgtctgttct ggctaagtca 2640
gacgccattt gagcagcgcc atattgtcga tggtttcagt tttgagttaa gcaaagtcgt 2700
tcgtccgtat attcgtgagc gcgttgttga ccagctggcg catattgatc tcactctggc 2760
ccaggcggtg gcgaaaaatc tcggtatcga actgactgac gaccagctga atatcacccc 2820
acctccggac gtcaacggtc tgaaaaagga tccatcctta agtttgtacg ccattcctga 2880
cggtgatgtg aaaggtcgcg tggtagcgat tttacttaat gatgaagtga gatcggcaga 2940
ccttctggcc attctcaagg cgctgaaggc caaaggcgtt catgccaaac tgctctactc 3000
ccgaatgggt gaagtgactg cggatgacgg tacggtgttg cctatagccg ctacctttgc 3060
cggtgcacct tcgctgacgg tcgatgcggt cattgtccct tgcggcaata tcgcggatat 3120
cgctgacaac ggcgatgcca actactacct gatggaagcc tacaaacacc ttaaaccgat 3180
tgcgctggcg ggtgacgcgc gcaagtttaa agcaacaatc aagatcgctg accagggtga 3240
agaagggatt gtggaagctg acagcgctga cggtagtttt atggatgaac tgctaacgct 3300
gatggcagca caccgcgtgt ggtcacgcat tcctaagatt gacaaaattc ctgcctgact 3360
cgagaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg 3420
ttgtttgtcg gtgaacgctc tccgagtagg acaaatccgc cgggagcgga tttgaacgtt 3480
gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa 3540
attaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttcctgtc 3600
gtcatatcta gcttattcag cgtttgtaca tatcgttaca cgctgaaacc aaccactcac 3660
ggaagtctgc catagggatt aatatagtta tttcaacggc cccgcagtgg ggttaaatga 3720
aaaaacaaat tgagggtatg acaatgaata tttcatctct ccgtaaagcg tttattttta 3780
tgggcgctgt agcggctttg tcactggtga acgcacaatc tgcgttggca gccaatgaat 3840
ccgctaaaga tatgacctgc caggaattta ttgatctgaa tccaaaagca atgaccccgg 3900
ttgcatggtg gatgctgcat gaagaaacag tatataaagg tggcgatacc gttactttaa 3960
atgaaaccga tctcactcaa attcctaaag tgatcgaata ctgtaagaaa aacccgcaga 4020
aaaatttgta taccttcaaa aatcaagcat ctaatgactt gccgaattaa ctcgagaggc 4080
atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 4140
cggtgaacgc tctccgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 4200
acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca 4260
gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg tcgtcatatc 4320
tagagtctcg agaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt 4380
tttatctgtt gtttgtcggt gaacgctctc cgagtaggac aaatccgccg ggagcggatt 4440
tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 4500
ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 4560
ttcctgtcgt catatcta 4578
<210> 77
<211> 5235
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 77
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgatttttct catgacgaaa gattcttttc ttttacaggg cttttggcag 180
ttgaaagata atcacgaaat gataaaaatc aattccctgt cagagatcaa aaaagtaggc 240
aataaaccct tcaaggttat cattgatacc tatcacaatc atatccttga tgaagaagcg 300
attaaatttc tggagaaatt agatgccgag agaattattg ttttggcacc ttatcacatc 360
agtaaactaa aagctaaagc gcctatttat tttgttagcc gcaaagaaag tatcaaaaat 420
cttcttgaga ttacttatgg taaacacttg ccccataaga attcacaatt atgtttttca 480
cataatcagt tcaaaattat gcaactgatt ctgaaaaata aaaatgaaag caatatcacg 540
tcgacgctca atatttcgca acaaacatta aagattcaga aattcaacat tatgtacaag 600
ctgaaactaa gacgtatgag cgacatcgtc accctgggta tcacatctta tttttagctc 660
gagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 720
tgtttgtcgg tgaacgctct ccgagtagga caaatccgcc gggagcggat ttgaacgttg 780
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 840
ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg 900
tcatatctag tccattcagc gtttgtacat atcgttacac gctgaaacca accactcacg 960
gaagtctgcc atgagccacg ctatagttat ttcaacggcc ccgcagtggg gttaaatgaa 1020
aaaacaaatt gagggtatga caatgtcatt cgaattacct gcactaccat atgctaaaga 1080
tgctctggca ccgcacattt ctgcggaaac catcgagtat cactacggca agcaccatca 1140
gacttatgtc actaacctga acaacctgat taaaggtacc gcgtttgaag gtaaatcact 1200
ggaagagatt attcgcagct ctgaaggtgg cgtattcaac aacgcagctc aggtctggaa 1260
ccatactttc tactggaact gcctggcacc gaacgccggt ggcgaaccga ctggaaaagt 1320
cgctgaagct atcgccgcat cttttggcag ctttgccgat ttcaaagcgc agtttactga 1380
tgcagcgatc aaaaactttg gttctggctg gacctggctg gtgaaaaaca gcgatggcaa 1440
actggctatc gtttcaacct ctaacgcggg tactccgctg accaccgatg cgactccgct 1500
gctgaccgtt gatgtctggg aacacgctta ttacatcgac tatcgcaatg cacgtcctgg 1560
ctatctggag cacttctggg cgctggtgaa ctgggaattc gtagcgaaaa atctcgctgc 1620
ataactcgag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1680
tatctgttgt ttgtcggtga acgctctccg agtaggacaa atccgccggg agcggatttg 1740
aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1800
catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1860
cctgtcgtca tatctaatct attcagcgtt tgtacatatc gttacacgct gaaaccaacc 1920
actcacggaa gtctgccata ctgaaaaata tagttatttc aacggccccg cagtggggtt 1980
aaatgaaaaa acaaattgag ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc 2040
agcaccagtc accactacac gattccagcg aagcgaaacc ggggatggac tcactggcac 2100
ctgaggacgg ctctcatcgt ccagcggctg aaccaacacc gccaggtgca caacctaccg 2160
ccccagggag cctgaaagcc cctgatacgc gtaacgaaaa acttaattct ctggaagacg 2220
tacgcaaagg cagtgaaaat tatgcgctga ccactaatca gggcgtgcgc atcgccgacg 2280
atcaaaactc actgcgtgcc ggtagccgtg gtccaacgct gctggaagat tttattctgc 2340
gcgagaaaat cacccacttt gaccatgagc gcattccgga acgtattgtt catgcacgcg 2400
gatcagccgc tcacggttat ttccagccat ataaaagctt aagcgatatt accaaagcgg 2460
atttcctctc agatccgaac aaaatcaccc cagtatttgt acgtttctct accgttcagg 2520
gtggtgctgg ctctgctgat accgtgcgtg atatccgtgg ctttgccacc aagttctata 2580
ccgaagaggg tatttttgac ctcgttggca ataacacgcc aatcttcttt atccaggatg 2640
cgcataaatt ccccgatttt gttcatgcgg taaaaccaga accgcactgg gcaattccac 2700
aagggcaaag tgcccacgat actttctggg attatgtttc tctgcaacct gaaactctgc 2760
acaacgtgat gtgggcgatg tcggatcgcg gcatcccccg cagttaccgc accatggaag 2820
gcttcggtat tcacaccttc cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt 2880
tccactggaa accactggca ggtaaagcct cactcgtttg ggatgaagca caaaaactca 2940
ccggacgtga cccggacttc caccgccgcg agttgtggga agccattgaa gcaggcgatt 3000
ttccggaata cgaactgggc ttccagttga ttcctgaaga agatgaattc aagttcgact 3060
tcgatcttct cgatccaacc aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg 3120
gcaaaatggt gctcaatcgc aacccggata acttctttgc tgaaaacgaa caggcggctt 3180
tccatcctgg gcatatcgtg ccgggactgg acttcaccaa cgatccgctg ttgcagggac 3240
gtttgttctc ctataccgat acacaaatca gtcgtcttgg tgggccgaat ttccatgaga 3300
ttccgattaa ccgtccgacc tgcccttacc ataatttcca gcgtgacggc atgcatcgca 3360
tggggatcga cactaacccg gcgaattacg aaccgaactc gattaacgat aactggccgc 3420
gcgaaacacc gccggggccg aaacgcggcg gttttgaatc ataccaggag cgcgtggaag 3480
gcaataaagt tcgcgagcgc agcccatcgt ttggcgaata ttattcccat ccgcgtctgt 3540
tctggctaag tcagacgcca tttgagcagc gccatattgt cgatggtttc agttttgagt 3600
taagcaaagt cgttcgtccg tatattcgtg agcgcgttgt tgaccagctg gcgcatattg 3660
atctcactct ggcccaggcg gtggcgaaaa atctcggtat cgaactgact gacgaccagc 3720
tgaatatcac cccacctccg gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt 3780
acgccattcc tgacggtgat gtgaaaggtc gcgtggtagc gattttactt aatgatgaag 3840
tgagatcggc agaccttctg gccattctca aggcgctgaa ggccaaaggc gttcatgcca 3900
aactgctcta ctcccgaatg ggtgaagtga ctgcggatga cggtacggtg ttgcctatag 3960
ccgctacctt tgccggtgca ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca 4020
atatcgcgga tatcgctgac aacggcgatg ccaactacta cctgatggaa gcctacaaac 4080
accttaaacc gattgcgctg gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg 4140
ctgaccaggg tgaagaaggg attgtggaag ctgacagcgc tgacggtagt tttatggatg 4200
aactgctaac gctgatggca gcacaccgcg tgtggtcacg cattcctaag attgacaaaa 4260
ttcctgcctg actcgagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 4320
ttcgttttat ctgttgtttg tcggtgaacg ctctccgagt aggacaaatc cgccgggagc 4380
ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 4440
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 4500
aactcttcct gtcgtcatat ctagcttatt cagcgtttgt acatatcgtt acacgctgaa 4560
accaaccact cacggaagtc tgccatcacg tgaagtatag ttatttcaac ggccccgcag 4620
tggggttaaa tgaaaaaaca aattgagggt atgacaatga atatttcatc tctccgtaaa 4680
gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca atctgcgttg 4740
gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct gaatccaaaa 4800
gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa aggtggcgat 4860
accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga atactgtaag 4920
aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga cttgccgaat 4980
taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 5040
atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga gcggatttga 5100
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 5160
atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc 5220
ctgtcgtcat atcta 5235
<210> 78
<211> 5235
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 78
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgatttttct catgacgaaa gattcttttc ttttacaggg cttttggcag 180
ttgaaagata atcacgaaat gataaaaatc aattccctgt cagagatcaa aaaagtaggc 240
aataaaccct tcaaggttat cattgatacc tatcacaatc atatccttga tgaagaagcg 300
attaaatttc tggagaaatt agatgccgag agaattattg ttttggcacc ttatcacatc 360
agtaaactaa aagctaaagc gcctatttat tttgttagcc gcaaagaaag tatcaaaaat 420
cttcttgaga ttacttatgg taaacacttg ccccataaga attcacaatt atgtttttca 480
cataatcagt tcaaaattat gcaactgatt ctgaaaaata aaaatgaaag caatatcacg 540
tcgacgctca atatttcgca acaaacatta aagattcaga aattcaacat tatgtacaag 600
ctgaaactaa gacgtatgag cgacatcgtc accctgggta tcacatctta tttttagctc 660
gagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 720
tgtttgtcgg tgaacgctct ccgagtagga caaatccgcc gggagcggat ttgaacgttg 780
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 840
ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg 900
tcatatctag tccattcagc gtttgtacat atcgttacac gctgaaacca accactcacg 960
gaagtctgcc atgagccacg ctatagttat ttcaacggcc ccgcagtggg gttaaatgaa 1020
aaaacaaatt gagggtatga caatgtcatt cgaattacct gcactaccat atgctaaaga 1080
tgctctggca ccgcacattt ctgcggaaac catcgagtat cactacggca agcaccatca 1140
gacttatgtc actaacctga acaacctgat taaaggtacc gcgtttgaag gtaaatcact 1200
ggaagagatt attcgcagct ctgaaggtgg cgtattcaac aacgcagctc aggtctggaa 1260
ccatactttc tactggaact gcctggcacc gaacgccggt ggcgaaccga ctggaaaagt 1320
cgctgaagct atcgccgcat cttttggcag ctttgccgat ttcaaagcgc agtttactga 1380
tgcagcgatc aaaaactttg gttctggctg gacctggctg gtgaaaaaca gcgatggcaa 1440
actggctatc gtttcaacct ctaacgcggg tactccgctg accaccgatg cgactccgct 1500
gctgaccgtt gatgtctggg aacacgctta ttacatcgac tatcgcaatg cacgtcctgg 1560
ctatctggag cacttctggg cgctggtgaa ctgggaattc gtagcgaaaa atctcgctgc 1620
ataactcgag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1680
tatctgttgt ttgtcggtga acgctctccg agtaggacaa atccgccggg agcggatttg 1740
aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1800
catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1860
cctgtcgtca tatctaatct attcagcgtt tgtacatatc gttacacgct gaaaccaacc 1920
actcacggaa gtctgccata ctgaaaaata tagttatttc aacggccccg cagtggggtt 1980
aaatgaaaaa acaaattgag ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc 2040
agcaccagtc accactacac gattccagcg aagcgaaacc ggggatggac tcactggcac 2100
ctgaggacgg ctctcatcgt ccagcggctg aaccaacacc gccaggtgca caacctaccg 2160
ccccagggag cctgaaagcc cctgatacgc gtaacgaaaa acttaattct ctggaagacg 2220
tacgcaaagg cagtgaaaat tatgcgctga ccactaatca gggcgtgcgc atcgccgacg 2280
atcaaaactc actgcgtgcc ggtagccgtg gtccaacgct gctggaagat tttattctgc 2340
gcgagaaaat cacccacttt gaccatgagc gcattccgga acgtattgtt catgcacgcg 2400
gatcagccgc tcacggttat ttccagccat ataaaagctt aagcgatatt accaaagcgg 2460
atttcctctc agatccgaac aaaatcaccc cagtatttgt acgtttctct accgttcagg 2520
gtggtgctgg ctctgctgat accgtgcgtg atatccgtgg ctttgccacc aagttctata 2580
ccgaagaggg tatttttgac ctcgttggca ataacacgcc aatcttcttt atccaggatg 2640
cgcataaatt ccccgatttt gttcatgcgg taaaaccaga accgcactgg gcaattccac 2700
aagggcaaag tgcccacgat actttctggg attatgtttc tctgcaacct gaaactctgc 2760
acaacgtgat gtgggcgatg tcggatcgcg gcatcccccg cagttaccgc accatggaag 2820
gcttcggtat tcacaccttc cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt 2880
tccactggaa accactggca ggtaaagcct cactcgtttg ggatgaagca caaaaactca 2940
ccggacgtga cccggacttc caccgccgcg agttgtggga agccattgaa gcaggcgatt 3000
ttccggaata cgaactgggc ttccagttga ttcctgaaga agatgaattc aagttcgact 3060
tcgatcttct cgatccaacc aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg 3120
gcaaaatggt gctcaatcgc aacccggata acttctttgc tgaaaacgaa caggcggctt 3180
tccatcctgg gcatatcgtg ccgggactgg acttcaccaa cgatccgctg ttgcagggac 3240
gtttgttctc ctataccgat acacaaatca gtcgtcttgg tgggccgaat ttccatgaga 3300
ttccgattaa ccgtccgacc tgcccttacc ataatttcca gcgtgacggc atgcatcgca 3360
tggggatcga cactaacccg gcgaattacg aaccgaactc gattaacgat aactggccgc 3420
gcgaaacacc gccggggccg aaacgcggcg gttttgaatc ataccaggag cgcgtggaag 3480
gcaataaagt tcgcgagcgc agcccatcgt ttggcgaata ttattcccat ccgcgtctgt 3540
tctggctaag tcagacgcca tttgagcagc gccatattgt cgatggtttc agttttgagt 3600
taagcaaagt cgttcgtccg tatattcgtg agcgcgttgt tgaccagctg gcgcatattg 3660
atctcactct ggcccaggcg gtggcgaaaa atctcggtat cgaactgact gacgaccagc 3720
tgaatatcac cccacctccg gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt 3780
acgccattcc tgacggtgat gtgaaaggtc gcgtggtagc gattttactt aatgatgaag 3840
tgagatcggc agaccttctg gccattctca aggcgctgaa ggccaaaggc gttcatgcca 3900
aactgctcta ctcccgaatg ggtgaagtga ctgcggatga cggtacggtg ttgcctatag 3960
ccgctacctt tgccggtgca ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca 4020
atatcgcgga tatcgctgac aacggcgatg ccaactacta cctgatggaa gcctacaaac 4080
accttaaacc gattgcgctg gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg 4140
ctgaccaggg tgaagaaggg attgtggaag ctgacagcgc tgacggtagt tttatggatg 4200
aactgctaac gctgatggca gcacaccgcg tgtggtcacg cattcctaag attgacaaaa 4260
ttcctgcctg actcgagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 4320
ttcgttttat ctgttgtttg tcggtgaacg ctctccgagt aggacaaatc cgccgggagc 4380
ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 4440
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 4500
aactcttcct gtcgtcatat ctagcttatt cagcgtttgt acatatcgtt acacgctgaa 4560
accaaccact cacggaagtc tgccatcacg tgaagtatag ttatttcaac ggccccgcag 4620
tggggttaaa tgaaaaaaca aattgagggt atgacaatga atatttcatc tctccgtaaa 4680
gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca atctgcgttg 4740
gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct gaatccaaaa 4800
gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa aggtggcgat 4860
accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga atactgtaag 4920
aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga cttgccgaat 4980
taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 5040
atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga gcggatttga 5100
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 5160
atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc 5220
ctgtcgtcat atcta 5235
<210> 79
<211> 5235
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 79
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccata 60
gggattaata tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgatttttct catgacgaaa gattcttttc ttttacaggg cttttggcag 180
ttgaaagata atcacgaaat gataaaaatc aattccctgt cagagatcaa aaaagtaggc 240
aataaaccct tcaaggttat cattgatacc tatcacaatc atatccttga tgaagaagcg 300
attaaatttc tggagaaatt agatgccgag agaattattg ttttggcacc ttatcacatc 360
agtaaactaa aagctaaagc gcctatttat tttgttagcc gcaaagaaag tatcaaaaat 420
cttcttgaga ttacttatgg taaacacttg ccccataaga attcacaatt atgtttttca 480
cataatcagt tcaaaattat gcaactgatt ctgaaaaata aaaatgaaag caatatcacg 540
tcgacgctca atatttcgca acaaacatta aagattcaga aattcaacat tatgtacaag 600
ctgaaactaa gacgtatgag cgacatcgtc accctgggta tcacatctta tttttagctc 660
gagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 720
tgtttgtcgg tgaacgctct ccgagtagga caaatccgcc gggagcggat ttgaacgttg 780
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 840
ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg 900
tcatatctag tccattcagc gtttgtacat atcgttacac gctgaaacca accactcacg 960
gaagtctgcc atagggatta atatagttat ttcaacggcc ccgcagtggg gttaaatgaa 1020
aaaacaaatt gagggtatga caatgtcatt cgaattacct gcactaccat atgctaaaga 1080
tgctctggca ccgcacattt ctgcggaaac catcgagtat cactacggca agcaccatca 1140
gacttatgtc actaacctga acaacctgat taaaggtacc gcgtttgaag gtaaatcact 1200
ggaagagatt attcgcagct ctgaaggtgg cgtattcaac aacgcagctc aggtctggaa 1260
ccatactttc tactggaact gcctggcacc gaacgccggt ggcgaaccga ctggaaaagt 1320
cgctgaagct atcgccgcat cttttggcag ctttgccgat ttcaaagcgc agtttactga 1380
tgcagcgatc aaaaactttg gttctggctg gacctggctg gtgaaaaaca gcgatggcaa 1440
actggctatc gtttcaacct ctaacgcggg tactccgctg accaccgatg cgactccgct 1500
gctgaccgtt gatgtctggg aacacgctta ttacatcgac tatcgcaatg cacgtcctgg 1560
ctatctggag cacttctggg cgctggtgaa ctgggaattc gtagcgaaaa atctcgctgc 1620
ataactcgag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1680
tatctgttgt ttgtcggtga acgctctccg agtaggacaa atccgccggg agcggatttg 1740
aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1800
catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1860
cctgtcgtca tatctaatct attcagcgtt tgtacatatc gttacacgct gaaaccaacc 1920
actcacggaa gtctgccatc acgtgaagta tagttatttc aacggccccg cagtggggtt 1980
aaatgaaaaa acaaattgag ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc 2040
agcaccagtc accactacac gattccagcg aagcgaaacc ggggatggac tcactggcac 2100
ctgaggacgg ctctcatcgt ccagcggctg aaccaacacc gccaggtgca caacctaccg 2160
ccccagggag cctgaaagcc cctgatacgc gtaacgaaaa acttaattct ctggaagacg 2220
tacgcaaagg cagtgaaaat tatgcgctga ccactaatca gggcgtgcgc atcgccgacg 2280
atcaaaactc actgcgtgcc ggtagccgtg gtccaacgct gctggaagat tttattctgc 2340
gcgagaaaat cacccacttt gaccatgagc gcattccgga acgtattgtt catgcacgcg 2400
gatcagccgc tcacggttat ttccagccat ataaaagctt aagcgatatt accaaagcgg 2460
atttcctctc agatccgaac aaaatcaccc cagtatttgt acgtttctct accgttcagg 2520
gtggtgctgg ctctgctgat accgtgcgtg atatccgtgg ctttgccacc aagttctata 2580
ccgaagaggg tatttttgac ctcgttggca ataacacgcc aatcttcttt atccaggatg 2640
cgcataaatt ccccgatttt gttcatgcgg taaaaccaga accgcactgg gcaattccac 2700
aagggcaaag tgcccacgat actttctggg attatgtttc tctgcaacct gaaactctgc 2760
acaacgtgat gtgggcgatg tcggatcgcg gcatcccccg cagttaccgc accatggaag 2820
gcttcggtat tcacaccttc cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt 2880
tccactggaa accactggca ggtaaagcct cactcgtttg ggatgaagca caaaaactca 2940
ccggacgtga cccggacttc caccgccgcg agttgtggga agccattgaa gcaggcgatt 3000
ttccggaata cgaactgggc ttccagttga ttcctgaaga agatgaattc aagttcgact 3060
tcgatcttct cgatccaacc aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg 3120
gcaaaatggt gctcaatcgc aacccggata acttctttgc tgaaaacgaa caggcggctt 3180
tccatcctgg gcatatcgtg ccgggactgg acttcaccaa cgatccgctg ttgcagggac 3240
gtttgttctc ctataccgat acacaaatca gtcgtcttgg tgggccgaat ttccatgaga 3300
ttccgattaa ccgtccgacc tgcccttacc ataatttcca gcgtgacggc atgcatcgca 3360
tggggatcga cactaacccg gcgaattacg aaccgaactc gattaacgat aactggccgc 3420
gcgaaacacc gccggggccg aaacgcggcg gttttgaatc ataccaggag cgcgtggaag 3480
gcaataaagt tcgcgagcgc agcccatcgt ttggcgaata ttattcccat ccgcgtctgt 3540
tctggctaag tcagacgcca tttgagcagc gccatattgt cgatggtttc agttttgagt 3600
taagcaaagt cgttcgtccg tatattcgtg agcgcgttgt tgaccagctg gcgcatattg 3660
atctcactct ggcccaggcg gtggcgaaaa atctcggtat cgaactgact gacgaccagc 3720
tgaatatcac cccacctccg gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt 3780
acgccattcc tgacggtgat gtgaaaggtc gcgtggtagc gattttactt aatgatgaag 3840
tgagatcggc agaccttctg gccattctca aggcgctgaa ggccaaaggc gttcatgcca 3900
aactgctcta ctcccgaatg ggtgaagtga ctgcggatga cggtacggtg ttgcctatag 3960
ccgctacctt tgccggtgca ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca 4020
atatcgcgga tatcgctgac aacggcgatg ccaactacta cctgatggaa gcctacaaac 4080
accttaaacc gattgcgctg gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg 4140
ctgaccaggg tgaagaaggg attgtggaag ctgacagcgc tgacggtagt tttatggatg 4200
aactgctaac gctgatggca gcacaccgcg tgtggtcacg cattcctaag attgacaaaa 4260
ttcctgcctg actcgagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 4320
ttcgttttat ctgttgtttg tcggtgaacg ctctccgagt aggacaaatc cgccgggagc 4380
ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 4440
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 4500
aactcttcct gtcgtcatat ctagcttatt cagcgtttgt acatatcgtt acacgctgaa 4560
accaaccact cacggaagtc tgccataggg attaatatag ttatttcaac ggccccgcag 4620
tggggttaaa tgaaaaaaca aattgagggt atgacaatga atatttcatc tctccgtaaa 4680
gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca atctgcgttg 4740
gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct gaatccaaaa 4800
gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa aggtggcgat 4860
accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga atactgtaag 4920
aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga cttgccgaat 4980
taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 5040
atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga gcggatttga 5100
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 5160
atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc 5220
ctgtcgtcat atcta 5235
<210> 80
<211> 5235
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 80
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatg 60
agccacgcta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgatttttct catgacgaaa gattcttttc ttttacaggg cttttggcag 180
ttgaaagata atcacgaaat gataaaaatc aattccctgt cagagatcaa aaaagtaggc 240
aataaaccct tcaaggttat cattgatacc tatcacaatc atatccttga tgaagaagcg 300
attaaatttc tggagaaatt agatgccgag agaattattg ttttggcacc ttatcacatc 360
agtaaactaa aagctaaagc gcctatttat tttgttagcc gcaaagaaag tatcaaaaat 420
cttcttgaga ttacttatgg taaacacttg ccccataaga attcacaatt atgtttttca 480
cataatcagt tcaaaattat gcaactgatt ctgaaaaata aaaatgaaag caatatcacg 540
tcgacgctca atatttcgca acaaacatta aagattcaga aattcaacat tatgtacaag 600
ctgaaactaa gacgtatgag cgacatcgtc accctgggta tcacatctta tttttagctc 660
gagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 720
tgtttgtcgg tgaacgctct ccgagtagga caaatccgcc gggagcggat ttgaacgttg 780
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 840
ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg 900
tcatatctag tccattcagc gtttgtacat atcgttacac gctgaaacca accactcacg 960
gaagtctgcc atgagccacg ctatagttat ttcaacggcc ccgcagtggg gttaaatgaa 1020
aaaacaaatt gagggtatga caatgtcatt cgaattacct gcactaccat atgctaaaga 1080
tgctctggca ccgcacattt ctgcggaaac catcgagtat cactacggca agcaccatca 1140
gacttatgtc actaacctga acaacctgat taaaggtacc gcgtttgaag gtaaatcact 1200
ggaagagatt attcgcagct ctgaaggtgg cgtattcaac aacgcagctc aggtctggaa 1260
ccatactttc tactggaact gcctggcacc gaacgccggt ggcgaaccga ctggaaaagt 1320
cgctgaagct atcgccgcat cttttggcag ctttgccgat ttcaaagcgc agtttactga 1380
tgcagcgatc aaaaactttg gttctggctg gacctggctg gtgaaaaaca gcgatggcaa 1440
actggctatc gtttcaacct ctaacgcggg tactccgctg accaccgatg cgactccgct 1500
gctgaccgtt gatgtctggg aacacgctta ttacatcgac tatcgcaatg cacgtcctgg 1560
ctatctggag cacttctggg cgctggtgaa ctgggaattc gtagcgaaaa atctcgctgc 1620
ataactcgag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1680
tatctgttgt ttgtcggtga acgctctccg agtaggacaa atccgccggg agcggatttg 1740
aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1800
catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1860
cctgtcgtca tatctaatct attcagcgtt tgtacatatc gttacacgct gaaaccaacc 1920
actcacggaa gtctgccatc acgtgaagta tagttatttc aacggccccg cagtggggtt 1980
aaatgaaaaa acaaattgag ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc 2040
agcaccagtc accactacac gattccagcg aagcgaaacc ggggatggac tcactggcac 2100
ctgaggacgg ctctcatcgt ccagcggctg aaccaacacc gccaggtgca caacctaccg 2160
ccccagggag cctgaaagcc cctgatacgc gtaacgaaaa acttaattct ctggaagacg 2220
tacgcaaagg cagtgaaaat tatgcgctga ccactaatca gggcgtgcgc atcgccgacg 2280
atcaaaactc actgcgtgcc ggtagccgtg gtccaacgct gctggaagat tttattctgc 2340
gcgagaaaat cacccacttt gaccatgagc gcattccgga acgtattgtt catgcacgcg 2400
gatcagccgc tcacggttat ttccagccat ataaaagctt aagcgatatt accaaagcgg 2460
atttcctctc agatccgaac aaaatcaccc cagtatttgt acgtttctct accgttcagg 2520
gtggtgctgg ctctgctgat accgtgcgtg atatccgtgg ctttgccacc aagttctata 2580
ccgaagaggg tatttttgac ctcgttggca ataacacgcc aatcttcttt atccaggatg 2640
cgcataaatt ccccgatttt gttcatgcgg taaaaccaga accgcactgg gcaattccac 2700
aagggcaaag tgcccacgat actttctggg attatgtttc tctgcaacct gaaactctgc 2760
acaacgtgat gtgggcgatg tcggatcgcg gcatcccccg cagttaccgc accatggaag 2820
gcttcggtat tcacaccttc cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt 2880
tccactggaa accactggca ggtaaagcct cactcgtttg ggatgaagca caaaaactca 2940
ccggacgtga cccggacttc caccgccgcg agttgtggga agccattgaa gcaggcgatt 3000
ttccggaata cgaactgggc ttccagttga ttcctgaaga agatgaattc aagttcgact 3060
tcgatcttct cgatccaacc aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg 3120
gcaaaatggt gctcaatcgc aacccggata acttctttgc tgaaaacgaa caggcggctt 3180
tccatcctgg gcatatcgtg ccgggactgg acttcaccaa cgatccgctg ttgcagggac 3240
gtttgttctc ctataccgat acacaaatca gtcgtcttgg tgggccgaat ttccatgaga 3300
ttccgattaa ccgtccgacc tgcccttacc ataatttcca gcgtgacggc atgcatcgca 3360
tggggatcga cactaacccg gcgaattacg aaccgaactc gattaacgat aactggccgc 3420
gcgaaacacc gccggggccg aaacgcggcg gttttgaatc ataccaggag cgcgtggaag 3480
gcaataaagt tcgcgagcgc agcccatcgt ttggcgaata ttattcccat ccgcgtctgt 3540
tctggctaag tcagacgcca tttgagcagc gccatattgt cgatggtttc agttttgagt 3600
taagcaaagt cgttcgtccg tatattcgtg agcgcgttgt tgaccagctg gcgcatattg 3660
atctcactct ggcccaggcg gtggcgaaaa atctcggtat cgaactgact gacgaccagc 3720
tgaatatcac cccacctccg gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt 3780
acgccattcc tgacggtgat gtgaaaggtc gcgtggtagc gattttactt aatgatgaag 3840
tgagatcggc agaccttctg gccattctca aggcgctgaa ggccaaaggc gttcatgcca 3900
aactgctcta ctcccgaatg ggtgaagtga ctgcggatga cggtacggtg ttgcctatag 3960
ccgctacctt tgccggtgca ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca 4020
atatcgcgga tatcgctgac aacggcgatg ccaactacta cctgatggaa gcctacaaac 4080
accttaaacc gattgcgctg gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg 4140
ctgaccaggg tgaagaaggg attgtggaag ctgacagcgc tgacggtagt tttatggatg 4200
aactgctaac gctgatggca gcacaccgcg tgtggtcacg cattcctaag attgacaaaa 4260
ttcctgcctg actcgagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 4320
ttcgttttat ctgttgtttg tcggtgaacg ctctccgagt aggacaaatc cgccgggagc 4380
ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 4440
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 4500
aactcttcct gtcgtcatat ctagcttatt cagcgtttgt acatatcgtt acacgctgaa 4560
accaaccact cacggaagtc tgccatcacg tgaagtatag ttatttcaac ggccccgcag 4620
tggggttaaa tgaaaaaaca aattgagggt atgacaatga atatttcatc tctccgtaaa 4680
gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca atctgcgttg 4740
gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct gaatccaaaa 4800
gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa aggtggcgat 4860
accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga atactgtaag 4920
aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga cttgccgaat 4980
taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 5040
atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga gcggatttga 5100
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 5160
atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc 5220
ctgtcgtcat atcta 5235
<210> 81
<211> 5235
<212> DNA
<213> Escherichia coli MG1655(Escherichia coli MG1655)
<400> 81
attcagcgtt tgtacatatc gttacacgct gaaaccaacc actcacggaa gtctgccatc 60
acgtgaagta tagttatttc aacggccccg cagtggggtt aaatgaaaaa acaaattgag 120
ggtatgacaa tgatttttct catgacgaaa gattcttttc ttttacaggg cttttggcag 180
ttgaaagata atcacgaaat gataaaaatc aattccctgt cagagatcaa aaaagtaggc 240
aataaaccct tcaaggttat cattgatacc tatcacaatc atatccttga tgaagaagcg 300
attaaatttc tggagaaatt agatgccgag agaattattg ttttggcacc ttatcacatc 360
agtaaactaa aagctaaagc gcctatttat tttgttagcc gcaaagaaag tatcaaaaat 420
cttcttgaga ttacttatgg taaacacttg ccccataaga attcacaatt atgtttttca 480
cataatcagt tcaaaattat gcaactgatt ctgaaaaata aaaatgaaag caatatcacg 540
tcgacgctca atatttcgca acaaacatta aagattcaga aattcaacat tatgtacaag 600
ctgaaactaa gacgtatgag cgacatcgtc accctgggta tcacatctta tttttagctc 660
gagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 720
tgtttgtcgg tgaacgctct ccgagtagga caaatccgcc gggagcggat ttgaacgttg 780
cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 840
ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttcctgtcg 900
tcatatctag tccattcagc gtttgtacat atcgttacac gctgaaacca accactcacg 960
gaagtctgcc atactgaaaa atatagttat ttcaacggcc ccgcagtggg gttaaatgaa 1020
aaaacaaatt gagggtatga caatgtcatt cgaattacct gcactaccat atgctaaaga 1080
tgctctggca ccgcacattt ctgcggaaac catcgagtat cactacggca agcaccatca 1140
gacttatgtc actaacctga acaacctgat taaaggtacc gcgtttgaag gtaaatcact 1200
ggaagagatt attcgcagct ctgaaggtgg cgtattcaac aacgcagctc aggtctggaa 1260
ccatactttc tactggaact gcctggcacc gaacgccggt ggcgaaccga ctggaaaagt 1320
cgctgaagct atcgccgcat cttttggcag ctttgccgat ttcaaagcgc agtttactga 1380
tgcagcgatc aaaaactttg gttctggctg gacctggctg gtgaaaaaca gcgatggcaa 1440
actggctatc gtttcaacct ctaacgcggg tactccgctg accaccgatg cgactccgct 1500
gctgaccgtt gatgtctggg aacacgctta ttacatcgac tatcgcaatg cacgtcctgg 1560
ctatctggag cacttctggg cgctggtgaa ctgggaattc gtagcgaaaa atctcgctgc 1620
ataactcgag aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 1680
tatctgttgt ttgtcggtga acgctctccg agtaggacaa atccgccggg agcggatttg 1740
aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 1800
catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt 1860
cctgtcgtca tatctaatct attcagcgtt tgtacatatc gttacacgct gaaaccaacc 1920
actcacggaa gtctgccata ctgaaaaata tagttatttc aacggccccg cagtggggtt 1980
aaatgaaaaa acaaattgag ggtatgacaa tgtcgcaaca taacgaaaag aacccacatc 2040
agcaccagtc accactacac gattccagcg aagcgaaacc ggggatggac tcactggcac 2100
ctgaggacgg ctctcatcgt ccagcggctg aaccaacacc gccaggtgca caacctaccg 2160
ccccagggag cctgaaagcc cctgatacgc gtaacgaaaa acttaattct ctggaagacg 2220
tacgcaaagg cagtgaaaat tatgcgctga ccactaatca gggcgtgcgc atcgccgacg 2280
atcaaaactc actgcgtgcc ggtagccgtg gtccaacgct gctggaagat tttattctgc 2340
gcgagaaaat cacccacttt gaccatgagc gcattccgga acgtattgtt catgcacgcg 2400
gatcagccgc tcacggttat ttccagccat ataaaagctt aagcgatatt accaaagcgg 2460
atttcctctc agatccgaac aaaatcaccc cagtatttgt acgtttctct accgttcagg 2520
gtggtgctgg ctctgctgat accgtgcgtg atatccgtgg ctttgccacc aagttctata 2580
ccgaagaggg tatttttgac ctcgttggca ataacacgcc aatcttcttt atccaggatg 2640
cgcataaatt ccccgatttt gttcatgcgg taaaaccaga accgcactgg gcaattccac 2700
aagggcaaag tgcccacgat actttctggg attatgtttc tctgcaacct gaaactctgc 2760
acaacgtgat gtgggcgatg tcggatcgcg gcatcccccg cagttaccgc accatggaag 2820
gcttcggtat tcacaccttc cgcctgatta atgccgaagg gaaggcaacg tttgtacgtt 2880
tccactggaa accactggca ggtaaagcct cactcgtttg ggatgaagca caaaaactca 2940
ccggacgtga cccggacttc caccgccgcg agttgtggga agccattgaa gcaggcgatt 3000
ttccggaata cgaactgggc ttccagttga ttcctgaaga agatgaattc aagttcgact 3060
tcgatcttct cgatccaacc aaacttatcc cggaagaact ggtgcccgtt cagcgtgtcg 3120
gcaaaatggt gctcaatcgc aacccggata acttctttgc tgaaaacgaa caggcggctt 3180
tccatcctgg gcatatcgtg ccgggactgg acttcaccaa cgatccgctg ttgcagggac 3240
gtttgttctc ctataccgat acacaaatca gtcgtcttgg tgggccgaat ttccatgaga 3300
ttccgattaa ccgtccgacc tgcccttacc ataatttcca gcgtgacggc atgcatcgca 3360
tggggatcga cactaacccg gcgaattacg aaccgaactc gattaacgat aactggccgc 3420
gcgaaacacc gccggggccg aaacgcggcg gttttgaatc ataccaggag cgcgtggaag 3480
gcaataaagt tcgcgagcgc agcccatcgt ttggcgaata ttattcccat ccgcgtctgt 3540
tctggctaag tcagacgcca tttgagcagc gccatattgt cgatggtttc agttttgagt 3600
taagcaaagt cgttcgtccg tatattcgtg agcgcgttgt tgaccagctg gcgcatattg 3660
atctcactct ggcccaggcg gtggcgaaaa atctcggtat cgaactgact gacgaccagc 3720
tgaatatcac cccacctccg gacgtcaacg gtctgaaaaa ggatccatcc ttaagtttgt 3780
acgccattcc tgacggtgat gtgaaaggtc gcgtggtagc gattttactt aatgatgaag 3840
tgagatcggc agaccttctg gccattctca aggcgctgaa ggccaaaggc gttcatgcca 3900
aactgctcta ctcccgaatg ggtgaagtga ctgcggatga cggtacggtg ttgcctatag 3960
ccgctacctt tgccggtgca ccttcgctga cggtcgatgc ggtcattgtc ccttgcggca 4020
atatcgcgga tatcgctgac aacggcgatg ccaactacta cctgatggaa gcctacaaac 4080
accttaaacc gattgcgctg gcgggtgacg cgcgcaagtt taaagcaaca atcaagatcg 4140
ctgaccaggg tgaagaaggg attgtggaag ctgacagcgc tgacggtagt tttatggatg 4200
aactgctaac gctgatggca gcacaccgcg tgtggtcacg cattcctaag attgacaaaa 4260
ttcctgcctg actcgagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 4320
ttcgttttat ctgttgtttg tcggtgaacg ctctccgagt aggacaaatc cgccgggagc 4380
ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac 4440
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca 4500
aactcttcct gtcgtcatat ctagcttatt cagcgtttgt acatatcgtt acacgctgaa 4560
accaaccact cacggaagtc tgccataggg attaatatag ttatttcaac ggccccgcag 4620
tggggttaaa tgaaaaaaca aattgagggt atgacaatga atatttcatc tctccgtaaa 4680
gcgtttattt ttatgggcgc tgtagcggct ttgtcactgg tgaacgcaca atctgcgttg 4740
gcagccaatg aatccgctaa agatatgacc tgccaggaat ttattgatct gaatccaaaa 4800
gcaatgaccc cggttgcatg gtggatgctg catgaagaaa cagtatataa aggtggcgat 4860
accgttactt taaatgaaac cgatctcact caaattccta aagtgatcga atactgtaag 4920
aaaaacccgc agaaaaattt gtataccttc aaaaatcaag catctaatga cttgccgaat 4980
taactcgaga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 5040
atctgttgtt tgtcggtgaa cgctctccga gtaggacaaa tccgccggga gcggatttga 5100
acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc 5160
atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttc 5220
ctgtcgtcat atcta 5235
<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
ctgctggcta ccctgtggaa 20
<210> 83
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
acacttccgt gagtggttgg tttcagnnnn nnnngatatg tacaaacgct g 51
<210> 84
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gaaaccaacc actcacggaa gtctgccatt cccaggatat agttatttca acggcccc 58
<210> 85
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ttccacaggg tagccagcag catc 24
<210> 86
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
caaattgagg gtatgacaat gatttttctc atgacgaaag 40
<210> 87
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
cctttcgttt tatttgatgc ctctcgagct aaaaataaga tgtgatac 48
<210> 88
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
caaattgagg gtatgacaat gtcattcgaa ttacctg 37
<210> 89
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
cctttcgttt tatttgatgc ctctcgagtt atgcagcgag atttttc 47
<210> 90
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
caaattgagg gtatgacaat gtcgcaacat aacgaaaag 39
<210> 91
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
cctttcgttt tatttgatgc ctctcgagtc aggcaggaat tttgtc 46
<210> 92
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ccaaattgag ggtatgacaa tgaatatttc atctctccg 39
<210> 93
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cctttcgttt tatttgatgc ctctcgagtt aattcggcaa gtcattag 48
<210> 94
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
gaccgggtcg aatttgcttt cg 22
<210> 95
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
tgtcataccc tcaatttgt 19
<210> 96
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ctcgagaggc atcaaataaa acg 23
<210> 97
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gaaattcgaa agcaaattcg acc 23
<210> 98
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
cgcaggtgtg gttatgctac tagagacggc cccgcagtgg ggtt 44
<210> 99
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
gcctttcgtt ttatttgatg cctctc 26
<210> 100
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
cgctagcgga gtgtatactg gcttactatg tt 32
<210> 101
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ctctagtagc ataaccacac ctgcgtcagg caggaatttt gtcaa 45
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
ctcgagaggc atcaaataaa acg 23
<210> 103
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
taagccagta tacactccgc tagc 24
<210> 104
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ggtctcaaat gattcagcgt ttgtacatat 30
<210> 105
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
ggtctcagga ctagatatga cgacaggaag 30
<210> 106
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ggtctcagtc cattcagcgt ttgtacatat 30
<210> 107
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
ggtctcaaga ttagatatga cgacaggaag 30
<210> 108
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ggtctcaatc tattcagcgt ttgtacatat 30
<210> 109
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ggtctcaact ctagatatga cgacaggaag 30
<210> 110
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
ggtctcagct tattcagcgt ttgtacatat 30
<210> 111
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ggtctcaact ctagatatga cgacaggaag 30

Claims (12)

1. An acid-resistant expression cassette, which is characterized by consisting of more than one promoter, more than one stress-resistant gene and more than one terminator, wherein the stress-resistant gene is selected from an acid-resistant system regulation transcription factor gene, an antioxidant protein gene and a periplasmic space chaperone protein gene, and the specific genes are a transcription factor gene gadE gene, an antioxidant protein gene sodB gene, a katE gene and a periplasmic space chaperone protein gene hdeB gene, and an acid response asr promoter library is constructed; the gadE gene code is an amino acid sequence shown as SEQ ID NO. 1; the sodB gene code is an amino acid sequence shown as SEQ ID NO. 2; the katE gene code is an amino acid sequence shown as SEQ ID NO. 3; the hdeB gene is coded by an amino acid sequence shown in SEQ ID NO. 4.
2. The acid-fast expression cassette of claim 1, wherein the promoter is a pH-responsive promoter;
the pH response promoter is asr promoter wild type or mutant thereof, and the gene code is shown in SEQ ID NO. 10-59;
the terminator is rrnB terminator;
the nucleotide sequence of the rrnB terminator is shown in SEQ ID NO 9.
3. The antacid expression cassette of claim 1, consisting of from 5 'to 3' a promoter selected from the group consisting of SEQ ID NO:11-13 and asr promoter mutants, the gadE gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 60-62.
4. The antacid expression cassette of claim 1, consisting of from 5 'to 3' a promoter mutant selected from the group consisting of SEQ ID NOS 11-13 and asr, the sodB gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 63-65.
5. The antacid expression cassette of claim 1, consisting of from 5 'to 3' a promoter mutant selected from the group consisting of SEQ ID NOS 11-13 and asr, the katE gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 66-68.
6. The antacid expression cassette of claim 1, consisting of from 5 'to 3' a promoter selected from the group consisting of mutants of the promoters of SEQ ID NOS: 11-13 and asr, the hdeB gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 69-71.
7. The antacid expression cassette of claim 1, consisting of from 5 'to 3' a promoter mutant selected from the group consisting of SEQ ID NOS: 11-13 and asr, the sodB gene, the katE gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 72-74.
8. The antacid expression cassette of claim 1, consisting of three parts, a first part comprising from 5 'to 3' asr promoter mutant, sodB gene and rrnB terminator, a second part comprising from 5 'to 3' asr promoter mutant, katE gene and rrnB terminator, and a third part comprising from 5 'to 3' asr promoter mutant, hdeB gene and rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 75 and 76.
9. The acid-resistant expression cassette of claim 1, which is composed of four parts, the first part comprising from 5 'to 3' the asr promoter mutant, the gadE gene, and the rrnB terminator, the second part comprising from 5 'to 3' the asr promoter mutant, the sodB gene, and the rrnB terminator, the third part comprising from 5 'to 3' the asr promoter mutant, the katE gene, and the rrnB terminator, and the fourth part comprising from 5 'to 3' the asr promoter mutant, the hdeB gene, and the rrnB terminator; the nucleotide sequence is shown in any one of SEQ ID NO 77-81.
10. Use of an antacid expression cassette according to claim 9, comprising the steps of:
(a) introducing an expression cassette of SEQ ID NO. 78-81 according to claim 9 into an organic acid-producing microorganism;
(b) fermenting the microorganism;
(c) the resulting organic acid is harvested.
11. The use of claim 10, wherein the organic acid comprises amino acids, succinic acid, citric acid, and lactic acid; the amino acid includes lysine, threonine, tryptophan or glutamic acid.
12. Method for high throughput screening of stress-resistant expression cassettes for microbial fermentation, characterized in that the method comprises the steps of:
(a) introducing an acid-resistant expression cassette gadE-sodB-katE-hdeB to be screened into the microorganism;
(b) evaluating the growth rate of the microorganisms under the stress pressure environment by using a high-throughput growth tester;
the is BioscreenC;
(c) evaluating the fermentation performance of the microorganism in a stress pressure environment by using a fermentation tank;
(d) identifying an expression cassette capable of increasing said survival rate and/or said growth rate and/or said fermentation performance of said microorganism as a stress resistant expression cassette for fermentation of a microorganism.
CN202111585143.XA 2021-12-20 2021-12-20 Acid-resistant expression cassette, application thereof and method for high-throughput screening of stress-resistant expression cassette for microbial fermentation Pending CN114410628A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150353973A1 (en) * 2012-08-20 2015-12-10 Evonik Degussa Gmbh Method for the fermentative production of l-amino acids using improved strains of the enterobacteriaceae family
CN105647912A (en) * 2014-12-01 2016-06-08 清华大学 Acid resistance expression cassette and screening method thereof
CN112359005A (en) * 2020-11-12 2021-02-12 江南大学 Escherichia coli engineering bacterium with improved acid stress capability and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150353973A1 (en) * 2012-08-20 2015-12-10 Evonik Degussa Gmbh Method for the fermentative production of l-amino acids using improved strains of the enterobacteriaceae family
CN105647912A (en) * 2014-12-01 2016-06-08 清华大学 Acid resistance expression cassette and screening method thereof
CN112359005A (en) * 2020-11-12 2021-02-12 江南大学 Escherichia coli engineering bacterium with improved acid stress capability and application thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALLISON HOYNES-O’CONNOR等: "Enabling Complex Genetic Circuits to Respond toExtrinsic Environmental Signals", BIOTECHNOLOGY AND BIOENGINEERING, vol. 114, no. 7, pages 1627 *
CHENGCHAO ZHU等: "Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli", BIOTECHNOLOGY AND BIOENGINEERING., pages 2018 *
DINGYU LIU等: "Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis", ACS SYNTH. BIOL., pages 1786 *
HEE JIN HWANG等: "Engineering and application of synthetic nar promoter for fne-tuning the expression of metabolic pathway genes in Escherichia coli", BIOTECHNOL BIOFUELS, vol. 11, pages 1 - 13 *
TINA BIRK等: "Acid stress response and protein induction in Campylobacter jejuni isolates with different acid tolerance", BMC MICROBIOLOGY, pages 11 *
丁秋露;: "大肠杆菌抗酸机制研究进展", 淮南师范学院学报, no. 05, pages 23 *

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