CN116445427A - Biosensor for responding to intracellular L-methionine concentration - Google Patents

Biosensor for responding to intracellular L-methionine concentration Download PDF

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CN116445427A
CN116445427A CN202210012050.6A CN202210012050A CN116445427A CN 116445427 A CN116445427 A CN 116445427A CN 202210012050 A CN202210012050 A CN 202210012050A CN 116445427 A CN116445427 A CN 116445427A
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methionine
biosensor
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张燕飞
黄建峰
蔡世杰
尤晓颜
赵国屏
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

A series of biosensors responding to intracellular L-methionine are constructed through gene recombination and protein directed evolution, the core element of the sensors is a methionyl-tRNA synthetase gene derived from escherichia coli and a wild-type promoter thereof, and the affinity of the methionyl-tRNA synthetase to L-methionine is reduced by remodelling an L-methionine binding pocket in the methionine-tRNA synthetase through the protein directed evolution. When the host cell contains only such mutant methionyl tRNA synthetase, only a sufficiently high concentration of L-methionine in the cell will ensure intracellular protein synthesis and normal growth of the cell. Therefore, a series of constructed biosensors can respond to different concentrations of L-methionine in living cells, and high-throughput screening of L-methionine high-yield strains can be realized through growth rate difference, so that the method has important application value in constructing the L-methionine high-yield strains.

Description

Biosensor for responding to intracellular L-methionine concentration
Technical Field
The invention belongs to the field of biochemical engineering, and particularly relates to construction and functional test of a biosensor for detecting the capacity of a strain to be detected in producing L-methionine.
Background
Methionine is an essential amino acid and is widely applied to the fields of feed, food processing, pharmacy and the like, and the annual demand is over 100 ten thousand tons. Germany win (Evonik) first achieved chemical synthesis of D, L-methionine in 1950s, a route which has now become the dominant production method for methionine. However, the chemical synthesis process of D, L-methionine needs to use highly toxic petroleum-based compounds such as acrolein, methyl mercaptan, hydrocyanic acid and the like as initial raw materials, the reaction process needs high temperature and high pressure, the environmental impact is large, and the requirements on equipment maintenance, production line site selection and management are severe. Under the conditions of environment, cost and the like, methionine self-sufficiency is not completely realized in China, and the imported dependence of 2020 is still as high as 80%.
Microorganisms can industrially produce numerous high-value-added chemicals by using clean, cheap and renewable raw materials, and are widely applied to the fields of feed, food, printing and dyeing, pharmacy and the like. However, the biosynthesis pathway of L-methionine in microorganisms is complex and lengthy and is subject to multi-level regulation, which results in slow development of the technology of breeding and fermentation industrialization of L-methionine high-yield strains. The synthesis of L-methionine can be effectively improved by optimizing the gene expression and the regulation and control release in the synthetic pathway through the conventional rational metabolic engineering design, but a great gap remains between industrial application, which suggests that the chassis bacteria need to be subjected to systematic design and reconstruction to realize breakthrough of fermentation titer. The conventional mutation breeding, such as gamma ray, ARTP (plasma mutagenesis at normal temperature and pressure), nitrosoguanidine and other physical and chemical means, is utilized to mutagenize chassis bacteria, and the generated microbial library is screened by methionine structural analogue (such as ethionine) flat plate, and is also confirmed by HPLC (high performance liquid chromatography) re-screening, so that no satisfactory effect is obtained. Although the physical and chemical mutagenesis can expand the mutation range and type to a certain extent, the strength of the primary screening mode of the structural analogue is limited, so that no small pressure is caused to the subsequent HPLC re-screening, and the breeding and transformation research of the high-yield strain cannot be effectively carried out. If a biosensor with adjustable screening strength and specific response to the concentration of L-methionine can be developed, the screening flux of a mutation library can be improved, and the method has positive effects on the directional breeding of high-yield strains of L-methionine.
Nurije mustfi et al reported a biosensor based on the transcription regulator Lrp that can respond to the intracellular L-methionine concentration of Corynebacterium glutamicum (The development and application of a single-cell biosensor for the detection of L-methionine and branched-chain amino acids.Metab.Eng.2012,14 (4): 449-57). Lrp proteins in Corynebacterium glutamicum activate expression of the branched amino acid transporter BrnFE when higher concentrations of L-methionine, L-leucine, L-valine and L-isoleucine are present in the cell. Cloning GFP gene downstream of Lrp response DNA sequence of brnFE gene and serially connecting Lrp expression cassette to construct a biosensor, which can sensitively respond to the concentration of L-methionine in corynebacterium glutamicum, and combining with FACS (flow cytometry fluorescence sorting technique) can effectively screen out L-methionine high-producing strain from random mutation library of corynebacterium glutamicum. However, this biosensor requires FACS assistance, has an upper limit on intracellular L-methionine concentration, and has poor biosensior response specificity and is also capable of responding to leucine, valine, and isoleucine.
Mohd Mohsin et al reported a biosensir (genetics-encoded nanosensor for quantitative monitoring of methionine in bacterial and yeast cells. Biosens. Bioelect. 2014.59:358-364) based on the FRET (fluorescence resonance energy transfer) principle that can respond to intracellular L-methionine concentrations. Fusion of CFP protein and YFP protein to MetN (methionine binding protein), respectivelyThe N-terminal and C-terminal, when L-methionine is combined with MetN, will generate conformational change, which provides for FRET of CFP and YFP at both ends, and can be expressed as change of concentration of intracellular L-methionine by 535/485nm fluorescence ratio. Further engineering of MetN, K for L-methionine d The value can be increased from 0.2mM to 2.4mM. This FRET-based biosensor has a good specificity and is equally effective in yeast, but the detection process is relatively complex and the cost is relatively high.
By applying the concept of synthetic biology, the development of a novel biosensor which can specifically respond to the concentration of intracellular L-methionine and cause the occurrence of obvious growth difference of high-yield strains and low-yield strains is beneficial to obviously reducing the screening cost of primary screening, reducing the screening pressure of secondary screening, effectively improving the screening flux of high-yield strains of L-methionine and has important significance for realizing the green fermentation production of L-methionine.
Disclosure of Invention
The object of the present invention is to provide a biosensor which can respond to the concentration of L-methionine in living cells, and which can respond to the concentration of L-methionine in living cells and cause the high-producing strain and the low-producing strain to exhibit a significant growth difference, thereby realizing high throughput screening of the L-methionine high-producing strain.
In order to achieve the above object, the present invention firstly provides a protein element of a biosensor for detecting L-methionine, characterized in that: is a truncated methionyl tRNA synthetase derived from escherichia coli or a mutant thereof, wherein the amino acid sequence of the truncated methionyl tRNA synthetase derived from escherichia coli is shown as SEQ ID NO.1 (Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthase.J.Bio.chem.1991.266:17136-17141); the mutant is a protein obtained by replacing at least one amino acid residue in the position L of 14, the position Y of 261 and the position H of 302 in the amino acid sequence shown in SEQ ID NO. 1.
Preferably, in the mutant, L at 14 th position in the amino acid sequence shown in SEQ ID NO.1 is replaced by K, G, V, D, Q, P, Y, C, F, A or W; the Y at 261 is replaced by V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F; the H at position 302 is replaced by M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N.
More preferably, the mutant is a double mutation in which L at position 14 in the amino acid sequence shown in SEQ ID NO.1 is replaced with L at position 14 as K, G, V, D, Q, P, Y, C, F, A or W, and Y at position 261 is replaced with V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F in any combination.
Further preferably, the mutant is a double mutant in which L at position 14 in the amino acid sequence shown in SEQ ID NO.1 is replaced with L at position 14 by K, G, V, D, Q, P, Y, C, F, A or W, and H at position 302 is replaced with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N in any combination.
It is another preferred that the mutant is a double mutant in which Y at position 261 in the amino acid sequence shown in SEQ ID NO.1 is replaced with V, R, C, G, S, I, K, M, H, A, T or F and H at position 302 is replaced with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N.
Further preferably, the mutant is a triple mutant in which L at position 14 in the amino acid sequence shown in SEQ ID NO.1 is replaced with L at position 14 as K, G, V, D, Q, P, Y, C, F, A or W, Y at position 261 is replaced with V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F, and H at position 302 is replaced with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N.
The invention thus provides a nucleic acid molecule encoding a protein element of the biosensor, in particular the nucleic acid molecule is DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA.
The invention further provides expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines comprising said nucleic acid molecules.
The invention also provides the use of a protein element of the biosensor, the nucleic acid molecule or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant microorganism or a transgenic cell line in: 1) Preparing a biosensor for detecting L-methionine; 2) Detecting the ability of the strain to produce L-methionine; 3) Preparing a biological material for producing L-methionine or L-methionine downstream products; 4) L-methionine is produced.
The invention further provides a biosensor for detecting L-methionine, which comprises an expression cassette, and is characterized in that the expression cassette comprises a wild-type promoter, an effector gene and a terminator from upstream to downstream in sequence; the effector gene is a nucleic acid molecule encoding the protein element, optionally further comprising a gene encoding a marker protein.
Preferably, the biosensor is a plasmid containing the expression cassette. The biosensor may be a plasmid containing the expression cassette. In one embodiment of the invention, the biosensor may specifically be a sensor plasmid pB2. In an embodiment of the present invention, the biosensor may specifically be any one of the sensor plasmids pB2-pB 122.
The invention further provides the use of the biosensor in accordance with the following aspects: detecting the ability of the strain to produce L-methionine; detecting the concentration of L-methionine in living cells of the strain; preparing a strain producing L-methionine or a downstream product of L-methionine; producing L-methionine; the starting microorganism of the biological material is escherichia coli, and more particularly the strain MG1655.
The invention has the beneficial effects that: the invention takes truncated methionyl-tRNA synthetase derived from escherichia coli as effector protein, and further changes the affinity of the truncated methionyl-tRNA synthetase to L-methionine through directed evolution, thus constructing a series of biosensors capable of sensing the intracellular L-methionine concentration. After the biosensor enters host bacteria with genome methionyl tRNA synthetase gene knocked out, when the concentration of L-methionine in the cell is low, the protein cannot be normally synthesized in the cell, and the growth speed of the cell is reduced; when intracellular L-methionine is high, intracellular protein synthesis is normal and cell growth rate is restored to normal. Thus, the concentration of L-methionine in the cells can be reflected by the growth rate of the cells on the agar plates. The series of sensors constructed by the invention can specifically respond to the concentration of intracellular L-methionine and cause obvious growth difference of high-yield strains and low-yield strains, can be helpful for obviously reducing the screening cost of primary screening, reducing the screening pressure of secondary screening, effectively improving the screening flux of high-yield strains and effective targets of L-methionine, and has important significance for realizing the green fermentation production of L-methionine.
Drawings
FIG. 1 is a schematic representation of the interaction pattern of a methionyl-tRNA synthetase with L-methionine in accordance with the invention;
FIG. 2 is a schematic diagram of a biosensor for evaluating intracellular L-methionine according to the present invention;
FIG. 3 shows the OD of the strain containing biosensor pB1 in example 3 600 Response to L-methionine concentration;
FIG. 4 shows the OD of the strain containing biosensor pB2 in example 4 600 Response to L-methionine concentration;
FIG. 5 is an OD of a strain containing biosensor pB2 in example 5 600 Response to addition of different concentrations of L-methionine;
FIG. 6 shows the OD of the strain pB2 containing the biosensor in example 6 600 Response specificity to different amino acids.
Detailed Description
The following examples merely illustrate the invention in further detail, but do not constitute any limitation thereof.
Reagents, materials and instruments used in the following examples and test examples are commercially available, except as specifically described; operations not specifically described are those conventional in the art.
Example 1 responsive L-methionine biosensor construction
1) Construction of a biosensor-initiating plasmid containing an E.coli-derived truncated Methanoaminoacyl-tRNA synthetase Gene
PCR amplified truncated methionyl tRNA synthetase gene and wild type promoter thereof
Genomic DNA of Escherichia coli MG1655 was extracted using a genomic extraction kit purchased from Promega company, and truncated methionyl-tRNA synthetase gene fragments containing a wild-type promoter in the genomic DNA were amplified by PCR using metGt-F and metGt-R as primers. The primer sequences are shown below:
metGt-F:TTTCTACTGAGCTGATTTTCTGCGCCCAAC；
metGt-R:GTCCATGTTATTCTTTAGAGGCTTCCACCAG。
PCR amplified vector backbone
The CDFD-ZH-F and CDFD-ZH-R are used as primers, the CDFD-lacZ-trc-yjeH plasmid with the nucleotide sequence shown as SEQ ID No.3 is used as a template, and the vector skeleton is amplified. The primers are as follows:
CDFD-ZH-F:CTCTAAAGAATAACATGGACTCGTCTACTAG
CDFD-ZH-R:GAAAATCAGCTCAGTAGAAAAGATCAAAGG
the two fragments were detected by agarose gel electrophoresis and then gel recovered using Axygen Gel Extraction Kit.
The two gene fragments obtained finally were ligated by ClonExpress II One Step Cloning Kit to finally form pB1 (the nucleotide sequence shown in SEQ ID No. 4).
E.coli Trans 5. Alpha. Competent cells were removed from the-80℃ultra-low temperature freezer and thawed in an ice bath. pB1 was added to competent cells, and the mixture was allowed to stand in an ice bath for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min, spreading 100 μl on a plate containing 50mg/L streptomycin sulfate, and culturing at 37 deg.C overnight. 10 colonies were picked and positive colonies were screened by colony PCR. 2 positive colonies were picked, cultured overnight, plasmids were extracted, and sent to company for sequencing. And finally obtaining the recombinant plasmid with correct sequence through sequence comparison.
2) Construction of a biosensor plasmid library containing E.coli-derived truncated Methanoaminoacyl-tRNA synthetase mutant genes
A library of truncated methionyl-tRNA synthetase mutants was constructed by site-directed mutagenesis using the pB1 plasmid as template and the primers listed in Table 1.
TABLE 1 site-directed mutagenesis primer
After completion of PCR, 9.75. Mu.L of the PCR product was taken and digested with 0.25. Mu.L of DpnI for 30min.
E.coli Trans 5. Alpha. Competent cells were removed from the-80℃ultra-low temperature freezer and thawed in an ice bath. 10. Mu.L of the DpnI digested PCR product was added to competent cells, and the mixture was allowed to stand in an ice bath for 30 minutes. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min, spreading 100 μl on plate containing 50mg/L streptomycin sulfate, and culturing at 37 deg.C overnight. 2 colonies were picked overnight, plasmids were extracted and sent to company for sequencing. And finally obtaining recombinant plasmids with correct sequences through sequence comparison, and constructing and obtaining a series of biosensor plasmids shown in Table 2.
TABLE 2 biosensor plasmid
Example 2.Escherichia coli MG1655 genomic methionyl-tRNA synthetase Gene (metG) knockout
a. Construction of the anaplerotic plasmid
metG belongs to an essential gene and cannot be directly knocked out, so that a complementing plasmid needs to be constructed first, and the survival of cells in the process of knocking out the genome metG is ensured.
Using Escherichia coli MG1655 genome DNA as template, P metG -F and P metG -R is a primer, PCR amplifying a truncated methionyl-tRNA synthetase gene fragment containing a wild type promoter in genomic DNA, the primer sequence being as follows:
P metG -F:taggaacttcGCTGATTTTCTGCGCCCAAC
P metG -R:GCGGCGGTTATTCTTTAGAGGCTTCCACCAG
the vector backbone was amplified using pTib1-F and pTib1-R as primers and the laboratory deposited pTib1 plasmid (nucleotide sequence shown in SEQ ID No. 5) as template. The primers are as follows:
pTib1-F:CTCTAAAGAATAAccgccgcagtctcacgc
pTib1-R:GAAAATCAGCGAAGTTCCTATTCTCTAGAAAG
the two fragments were detected by agarose gel electrophoresis and then gel recovered using Axygen Gel Extraction Kit.
The two gene fragments obtained finally were ligated by ClonExpress II One Step Cloning Kit to finally form pR1 (the nucleotide sequence is shown in SEQ ID No. 6).
E.coli Trans 5. Alpha. Competent cells were removed from the-80℃ultra-low temperature freezer and thawed in an ice bath. pR1 was added to competent cells and allowed to stand in an ice bath for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min, spreading 100 μl on a plate containing 50mg/L chloramphenicol, and culturing at 37 deg.C overnight in an inverted manner. 10 colonies were picked and positive colonies were screened by colony PCR. 2 positive colonies were picked, cultured overnight, plasmids were extracted, and sent to company for sequencing. And finally obtaining the recombinant plasmid with correct sequence through sequence comparison.
b. Genome metG gene knockout
The first step: N20-F and N20-R amplifications using pTargetF (Addgene # 62226) plasmid as template
The sgRNA fragment was used to construct pTarget-metG with the following primer sequences:
N20-F：TCAAGTCGCGAAGAAAATTCgttttagagctagaaatagc
N20-R：GAATTTTCTTCGCGACTTGAactagtattatacctaggac
10. Mu.L of the PCR product was digested with 0.25. Mu.L of DpnI enzyme for 30min.
E.coli Trans 5. Alpha. Competent cells were removed from the-80℃ultra-low temperature freezer and thawed in an ice bath. pTarget-metG was added to competent cells and the mixture was allowed to stand in an ice bath for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min, and applying 100 μl onto plate containing 50mg/L spectinomycin, and culturing at 37 deg.C overnight in an inverted manner. 2 colonies were picked overnight, plasmids were extracted and sent to company for sequencing. And finally obtaining the recombinant plasmid with correct sequence through sequence comparison.
And a second step of: using Escherichia coli MG1655 genome DNA as a template, amplifying upstream homology arm fragments by Udon-F and Udon-R respectively, and amplifying downstream homology arm fragments by Ddon-F and Ddon-R; the pTarget-metG plasmid obtained in the first step was used as a template, and pTG-VF and pTG-VR were used to amplify the plasmid backbone to construct pTarget-metG-donor. The primer sequences were as follows:
Udon-F：ctttttttgaGGACGATTTACCCACGCCGC
Udon-R：AGGGGGATTACATAGTAGGCATTACTTCTTA
Ddon-F：GCCTACTATGTAATCCCCCTTCAAGGCGCTG
Ddon-R：gcaggtcgacAATATCTTTCGCCAGCCACG
pTG-VF：GAAAGATATTgtcgacctgcagaagcttag
pTG-VR：TAAATCGTCCtcaaaaaaagcaccgactcg
and a third step of: amplifying metG gene fragments which do not contain corresponding PAM sites in pTarget-metG-donor by using Escherichia coli MG1655 genome DNA as a template and pTGd-metG-F and pTGd-metG-R as primers; the vector backbone was amplified using pTarget-metG-donor as templates, pTGd-F and pTGd-F as templates. Construction of pTarget-metG-donor-metG, primer sequences were as follows:
pTGd-metG-F:AATGCCTACTATGACTCAAGTCGCGAAGAAAATTCTAGTGACGTGCGCAC
pTGd-metG-R:GTTTATGCTAGAGCGGCATCTTATTTCACC
pTGd-F:GATGCCGCTCTCGCATAAACCTGATTGATT
pTGd-R:CTTGAGTCATAGTAGGCATTACTTCTTAAT
each of the above fragments was subjected to agarose gel electrophoresis and then subjected to gel recovery using Axygen Gel Extraction Kit.
The finally obtained gene fragment was ligated by using ClonExpress II One Step Cloning Kit to finally form pTarget-metG-donor-metG (nucleotide sequence shown as SEQ ID No. 7).
E.coli Trans 5. Alpha. Competent cells were removed from the-80℃ultra-low temperature freezer and thawed in an ice bath. pTarget-metG-donor-metG was added to competent cells and allowed to stand in ice for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min, and applying 100 μl onto plate containing 50mg/L spectinomycin, and culturing at 37 deg.C overnight in an inverted manner. 10 colonies were picked and positive colonies were screened by colony PCR. 2 colonies were picked overnight, plasmids were extracted and sent to company for sequencing. And finally obtaining the recombinant plasmid with correct sequence through sequence comparison.
Fourth, reference (Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Appl Environ microbiol 2015) performed genomic metG knockout. The difference is that: after pCas and pTarget-metG-donor-metG co-operate to complete genome metG knockout to obtain e.coli MG1655 Δmetg/(pcas+ptarget-metG-donor-metG), a make-up plasmid pR1 is transformed into the strain to construct e.coli MG1655 Δmetg/(pcas+ptarget-metG-donor-metg+pr1), and then normal plasmid elimination steps are entered to sequentially eliminate pTarget-metG-donor-metG and pCas, finally the e.coli MG1655 Δmetg/pR1 is constructed.
Example 3 functional test of sensor plasmid pB1
E.coliMG1655. DELTA. MetG/pR1 competence was removed from the-80℃ultra-low temperature refrigerator and thawed in an ice bath. Sensor plasmid pB1 was added to the senseIn the cells in the state, the ice bath was left for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min. All resuscitated products were transferred to LB tubes (additional L-methionine to final concentration of 0.8 g/L) containing 50mg/L streptomycin sulfate and incubated overnight at 42℃at 220 rpm. The overnight cultures were subjected to 10 5 Single colonies were isolated by dilution with LB agarose plates containing 50mg/L streptomycin sulfate. The chloramphenicol resistance of the single colony was checked, 2 single clones with resistance to chloramphenicol eliminated were picked and incubated overnight at 37℃to extract plasmids, and sent to the company for sequencing. And finally obtaining recombinant bacteria E.coli MG1655 delta metG/pB1 with correct sequence through sequence comparison.
E.coli MG1655. Delta. MetG/pB1 seed solution was inoculated into M9 medium and M9 medium containing 0.8g/L L-methionine, respectively, and cultured with shaking at 37℃and 220rpm, and OD was measured every 3 hours 600 . The detection result is shown in FIG. 3. As shown, exogenous addition of L-methionine did not affect the growth of recombinant E.coli MG1655. Delta. MetG/pB1, and there was no significant difference in growth rate between the control group (no addition of L-methionine) and the experimental group (addition of 0.8g/L L-methionine). The above results indicate that the sensor plasmid pB1 (containing a truncated wild-type methionyl-tRNA synthetase gene) is not responsive to L-methionine concentration and does not correlate the growth rate of host E.coli MG1655. Delta. MetG with intracellular L-methionine concentration.
Example 4 functional test of sensor plasmid pB2
E.coli MG1655. Delta. MetG/pR1 competence was taken out from the-80℃ultra-low temperature refrigerator and thawed in an ice bath. The sensor plasmid pB2 (in which L at position 14 of the methionyl tRNA synthetase is mutated to K) was added to competent cells and allowed to stand in ice for 30min. Heat-shocking at 42 deg.C for 90s, ice-bathing for 5min, adding 700 μl of antibiotic-free LB medium, shaking at 37 deg.C and 220rpm for resuscitation for 60min. All resuscitated products were transferred to LB tubes (additional L-methionine to final concentration of 0.8 g/L) containing 50mg/L streptomycin sulfate and incubated overnight at 42℃at 220 rpm. The overnight cultures were subjected to 10 5 Single colonies were isolated by dilution with LB agarose plates containing 50mg/L streptomycin sulfate. Chloramphenicol resistance of single colony was checked, 2 Chloromycetes were selectedThe monoclonal with the resistance removed was incubated overnight at 37℃to extract plasmids, and sent to the company for sequencing. And finally obtaining recombinant bacteria E.coli MG1655 delta metG/pB2 with correct sequence through sequence comparison.
E.coli MG1655. Delta. MetG/pB2 seed solution was inoculated into M9 medium and M9 medium containing 0.8g/L L-methionine, respectively, and cultured with shaking at 37℃and 220rpm, and OD was measured every 3 hours 600 . The detection results are shown in FIG. 4. As shown, when L-methionine was not exogenously added, E.coli MG1655. Delta. MetG/pB2 growth was almost arrested; and when L-methionine was exogenously added to 0.8g/L of L-methionine, E.coli MG1655. Delta. MetG/pB2 was grown completely restored to normal. The above results indicate that the sensor plasmid pB2 (containing the truncated methionyl-tRNA synthetase mutant L14K) senses L-methionine concentration and makes the growth rate of the host E.coli MG1655. Delta. MetG show a strong correlation with intracellular L-methionine concentration. Thus, sensor pB2 can be used to screen E.coli MG1655. Delta. MetG mutant strain for high yield of L-methionine.
Example 5 concentration response Range test of sensor plasmid pB2 to L-methionine
Inoculating E.coli MG1655 delta. MetG/pB2 seed solution into M9 medium containing 0.001g/L, 0.01g/L and 0.1g/L L-methionine, shake culturing at 37deg.C and 220rpm, and measuring OD every 3 hr 600 . The detection results are shown in FIG. 5. As shown, when L-methionine was exogenously added at various concentrations, the E.coli MG1655. Delta. MetG/pB2 growth was affected to varying degrees. When 0.001g/L L-methionine was exogenously added, little was possible to restore E.coli MG1655 ΔmetG/pB2 arrested growth; e.coli MG1655. Delta. MetG/pB2 growth was slightly restored with the addition of 0.01g/L L-methionine, but OD after 12h of incubation 600 The values were still significantly lower than E.coli MG1655 ΔmetG/pB1 (FIG. 3); e.coli MG1655. Delta. MetG/pB2 growth was substantially restored when 0.1g/L L-methionine was added. Thus, E.coli MG1655. Delta. MetG/pB2 had a response concentration of L-methionine to external addition in the range of 0.01-0.1g/L.
Example 6 response specificity test of sensor plasmid pB2 to L-methionine
methionyl-tRNA synthase vs. ammoniaThe selectivity of the base acid influences the constitution of the cell proteome, so that the ultra-high fidelity of the screened functional biosensor pBn (pBn corresponds to pB2 to pB122 in the foregoing) to the amino acids should not be changed. To verify the above, E.coli MG1655. Delta. MetG/pB2 seed solutions were inoculated into M9 medium containing 0.5g/L L-amino acid, respectively, and cultured at 37℃under shaking at 220rpm for 12 hours, and OD was measured 600 。
The detection results are shown in FIG. 6. As shown, exogenous addition of 0.5g/L L-methionine can restore E.coli MG1655 ΔmetG/pB2 growth; exogenous addition of L-glycine and L-arginine can promote E.coli MG1655. Delta. MetG/pB2 growth to some extent, but OD 600 Significantly lower than the L-methionine addition group; the remaining 16 amino acid additions were not significantly different from the control (control) and did not promote E.coli MG1655 ΔmetG/pB2 to resume growth. The above results indicate that E.coli MG1655. Delta. MetG/pB2 growth only gave a specific response to L-methionine addition.
Example 7 characterization of growth coupling Effect of other efficient sensor plasmids
A series of strains E.coli MG1655. Delta. MetG/pBn (pBn corresponds to pB2-pB122 hereinabove) were constructed in accordance with the methods described in examples 4 and 5 and tested for correlation between growth and exogenous L-methionine addition concentration. By exogenously adding different concentrations of L-methionine (0,0.00001,0.0001,0.001,0.01 and 0.1 g/L), a series of potent growth coupling effects of the biosensor plasmid were characterized. As shown in Table 3, the "L-methionine response concentration" means the L-methionine addition concentration at the time of complete growth recovery, and all the biosensor plasmids responded to L-methionine to varying degrees, at a minimum of 0.0001g/L and at most 0.1g/L. The above shows that a series of constructed and obtained sensor plasmids pB2-pB122 can sense the concentration of intracellular L-methionine and have different sensing intensities, and L-methionine high-producing strains can be gradually screened out by gradually adjusting the screening intensity.
TABLE 3 response concentration of different sensor plasmids to L-methionine
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Thus, the L-methionine biosensor constructed by the present invention can screen the L-methionine production capacity of the strain with high throughput; the target strain can be modified accordingly to obtain a strain with high yield of the downstream product of L-methionine (such as S-adenosyl-L-methionine). The invention has important application value in strain transformation and metabolic engineering.
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> A biosensor for responding to intracellular L-methionine concentration
<130> 2021.10.26
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 549
<212> PRT
<213> Escherichia coli
<400> 1
Met Thr Gln Val Ala Lys Lys Ile Leu Val Thr Cys Ala Leu Pro Tyr
Ala Asn Gly Ser Ile His Leu Gly His Met Leu Glu His Ile Gln Ala
Asp Val Trp Val Arg Tyr Gln Arg Met Arg Gly His Glu Val Asn Phe
Ile Cys Ala Asp Asp Ala His Gly Thr Pro Ile Met Leu Lys Ala Gln
Gln Leu Gly Ile Thr Pro Glu Gln Met Ile Gly Glu Met Ser Gln Glu
His Gln Thr Asp Phe Ala Gly Phe Asn Ile Ser Tyr Asp Asn Tyr His
Ser Thr His Ser Glu Glu Asn Arg Gln Leu Ser Glu Leu Ile Tyr Ser
Arg Leu Lys Glu Asn Gly Phe Ile Lys Asn Arg Thr Ile Ser Gln Leu
Tyr Asp Pro Glu Lys Gly Met Phe Leu Pro Asp Arg Phe Val Lys Gly
Thr Cys Pro Lys Cys Lys Ser Pro Asp Gln Tyr Gly Asp Asn Cys Glu
Val Cys Gly Ala Thr Tyr Ser Pro Thr Glu Leu Ile Glu Pro Lys Ser
Val Val Ser Gly Ala Thr Pro Val Met Arg Asp Ser Glu His Phe Phe
Phe Asp Leu Pro Ser Phe Ser Glu Met Leu Gln Ala Trp Thr Arg Ser
Gly Ala Leu Gln Glu Gln Val Ala Asn Lys Met Gln Glu Trp Phe Glu
Ser Gly Leu Gln Gln Trp Asp Ile Ser Arg Asp Ala Pro Tyr Phe Gly
Phe Glu Ile Pro Asn Ala Pro Gly Lys Tyr Phe Tyr Val Trp Leu Asp
Ala Pro Ile Gly Tyr Met Gly Ser Phe Lys Asn Leu Cys Asp Lys Arg
Gly Asp Ser Val Ser Phe Asp Glu Tyr Trp Lys Lys Asp Ser Thr Ala
Glu Leu Tyr His Phe Ile Gly Lys Asp Ile Val Tyr Phe His Ser Leu
Phe Trp Pro Ala Met Leu Glu Gly Ser Asn Phe Arg Lys Pro Ser Asn
Leu Phe Val His Gly Tyr Val Thr Val Asn Gly Ala Lys Met Ser Lys
Ser Arg Gly Thr Phe Ile Lys Ala Ser Thr Trp Leu Asn His Phe Asp
Ala Asp Ser Leu Arg Tyr Tyr Tyr Thr Ala Lys Leu Ser Ser Arg Ile
Asp Asp Ile Asp Leu Asn Leu Glu Asp Phe Val Gln Arg Val Asn Ala
Asp Ile Val Asn Lys Val Val Asn Leu Ala Ser Arg Asn Ala Gly Phe
Ile Asn Lys Arg Phe Asp Gly Val Leu Ala Ser Glu Leu Ala Asp Pro
Gln Leu Tyr Lys Thr Phe Thr Asp Ala Ala Glu Val Ile Gly Glu Ala
Trp Glu Ser Arg Glu Phe Gly Lys Ala Val Arg Glu Ile Met Ala Leu
Ala Asp Leu Ala Asn Arg Tyr Val Asp Glu Gln Ala Pro Trp Val Val
Ala Lys Gln Glu Gly Arg Asp Ala Asp Leu Gln Ala Ile Cys Ser Met
Gly Ile Asn Leu Phe Arg Val Leu Met Thr Tyr Leu Lys Pro Val Leu
Pro Lys Leu Thr Glu Arg Ala Glu Ala Phe Leu Asn Thr Glu Leu Thr
Trp Asp Gly Ile Gln Gln Pro Leu Leu Gly His Lys Val Asn Pro Phe
Lys Ala Leu Tyr Asn Arg Ile Asp Met Arg Gln Val Glu Ala Leu Val
Glu Ala Ser Lys Glu
<210> 2
<211> 612
<212> DNA
<213> Escherichia coli
<400> 2
gctgattttc tgcgcccaac attgttggga ttgatgggcc gtagatatcg gcgtccagaa 60
tgccgacttt cgccccttct gccgccagcg ccagtgccag gtttaccgcc gtggacgatt 120
tacccacgcc gcctttaccg gaactcaccg cgataatgtt cttcacaccg ttaattcccg 180
gctgattctt cacgcgtttc agagtggcta tgttatgcga cagtttccag tcgatagctt 240
tcgccccggt gatgcgcagc agttcggcgc tacattgctc ttttaactct tcgaacgcgc 300
tatgccagac aaacggcata accagttcca catgcaacgt gtcgtccatc caggcgacat 360
ggtgcaacgc tttcagcgtg gtgaggttat gcttcagggt tgggtgctga aaattggcca 420
gtgtcccggc gaccattgcg cgcaaggctt ctggtgattt ggcctgggat tgttcgttca 480
tcccgactcc ttttttgtat agataaacca tcagctgata gtttacctga agaatataga 540
gaagtactta cttaacattt tcccatttgg tactatctaa ccccttttca ctattaagaa 600
gtaatgccta ct 612
<210> 3
<211> 6511
<212> DNA
<213> Synthesis
<400> 3
gcgctgcgga cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg 60
tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca 120
gagttcacat aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg 180
aatctgacag tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt 240
cgctccctct tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt 300
tctcccttac gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt 360
gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg 420
cgccttatcc ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact 480
ggcagcagcc attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg 540
ctcttgaagt gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc 600
tgcgaaagcc agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc 660
acctccccag gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc 720
tcaagaagat cctttgatct tttctactga taacatggac tcgtctacta gcgcagctga 780
gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 840
gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 900
ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 960
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 1020
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 1080
gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc 1140
ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca 1200
tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc 1260
cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga 1320
cgcgaattat ttttgatggc gttaactcgg cgtttcatct gtggtgcaac gggcgctggg 1380
tcggttacgg ccaggacagt cgtttgccgt ctgaatttga cctgagcgca tttttacgcg 1440
ccggagaaaa ccgcctcgcg gtgatggtgc tgcgctggag tgacggcagt tatctggaag 1500
atcaggatat gtggcggatg agcggcattt tccgtgacgt ctcgttgctg cataaaccga 1560
ctacacaaat cagcgatttc catgttgcca ctcgctttaa tgatgatttc agccgcgctg 1620
tactggaggc tgaagttcag atgtgcggcg agttgcgtga ctacctacgg gtaacagttt 1680
ctttatggca gggtgaaacg caggtcgcca gcggcaccgc gcctttcggc ggtgaaatta 1740
tcgatgagcg tggtggttat gccgatcgcg tcacactacg tctgaacgtc gaaaacccga 1800
aactgtggag cgccgaaatc ccgaatctct atcgtgcggt ggttgaactg cacaccgccg 1860
acggcacgct gattgaagca gaagcctgcg atgtcggttt ccgcgaggtg cggattgaaa 1920
atggtctgct gctgctgaac ggcaagccgt tgctgattcg aggcgttaac cgtcacgagc 1980
atcatcctct gcatggtcag gtcatggatg agcagacgat ggtgcaggat atcctgctga 2040
tgaagcagaa caactttaac gccgtgcgct gttcgcatta tccgaaccat ccgctgtggt 2100
acacgctgtg cgaccgctac ggcctgtatg tggtggatga agccaatatt gaaacccacg 2160
gcatggtgcc aatgaatcgt ctgaccgatg atccgcgctg gctaccggcg atgagcgaac 2220
gcgtaacgcg aatggtgcag cgcgatcgta atcacccgag tgtgatcatc tggtcgctgg 2280
ggaatgaatc aggccacggc gctaatcacg acgcgctgta tcgctggatc aaatctgtcg 2340
atccttcccg cccggtgcag tatgaaggcg gcggagccga caccacggcc accgatatta 2400
tttgcccgat gtacgcgcgc gtggatgaag accagccctt cccggctgtg ccgaaatggt 2460
ccatcaaaaa atggctttcg ctacctggag agacgcgccc gctgatcctt tgcgaatacg 2520
cccacgcgat gggtaacagt cttggcggtt tcgctaaata ctggcaggcg tttcgtcagt 2580
atccccgttt acagggcggc ttcgtctggg actgggtgga tcagtcgctg attaaatatg 2640
atgaaaacgg caacccgtgg tcggcttacg gcggtgattt tggcgatacg ccgaacgatc 2700
gccagttctg tatgaacggt ctggtctttg ccgaccgcac gccgcatcca gcgctgacgg 2760
aagcaaaaca ccagcagcag tttttccagt tccgtttatc cgggcaaacc atcgaagtga 2820
ccagcgaata cctgttccgt catagcgata acgagctcct gcactggatg gtggcgctgg 2880
atggtaagcc gctggcaagc ggtgaagtgc ctctggatgt cgctccacaa ggtaaacagt 2940
tgattgaact gcctgaacta ccgcagccgg agagcgccgg gcaactctgg ctcacagtac 3000
gcgtagtgca accgaacgcg accgcatggt cagaagccgg gcacatcagc gcctggcagc 3060
agtggcgtct ggcggaaaac ctcagtgtga cgctccccgc cgcgtcccac gccatcccgc 3120
atctgaccac cagcgaaatg gatttttgca tcgagctggg taataagcgt tggcaattta 3180
accgccagtc aggctttctt tcacagatgt ggattggcga taaaaaacaa ctgctgacgc 3240
cgctgcgcga tcagttcacc cgtgcaccgc tggataacga cattggcgta agtgaagcga 3300
cccgcattga ccctaacgcc tgggtcgaac gctggaaggc ggcgggccat taccaggccg 3360
aagcagcgtt gttgcagtgc acggcagata cacttgctga tgcggtgctg attacgaccg 3420
ctcacgcgtg gcagcatcag gggaaaacct tatttatcag ccggaaaacc taccggattg 3480
atggtagtgg tcaaatggcg attaccgttg atgttgaagt ggcgagcgat acaccgcatc 3540
cggcgcggat tggcctgaac tgccagctgg cgcaggtagc agagcgggta aactggctcg 3600
gattagggcc gcaagaaaac tatcccgacc gccttactgc cgcctgtttt gaccgctggg 3660
atctgccatt gtcagacatg tataccccgt acgtcttccc gagcgaaaac ggtctgcgct 3720
gcgggacgcg cgaattgaat tatggcccac accagtggcg cggcgacttc cagttcaaca 3780
tcagccgcta cagtcaacag caactgatgg aaaccagcca tcgccatctg ctgcacgcgg 3840
aagaaggcac atggctgaat atcgacggtt tccatatggg gattggtggc gacgactcct 3900
ggagcccgtc agtatcggcg gaattccagc tgagcgccgg tcgctaccat taccagttgg 3960
tctggtgtca aaaataatta attaaggaat tgtgagcgga taacaatttc acacaggaaa 4020
cagaccccta ggctgctgcc attgacaatt aatcatccgg ctcgtataat gtgtcacaca 4080
ggaaacagac catgagtgga ctcaaacaag aactggggct ggcccagggc attggcctgc 4140
tatcgacgtc attattaggc actggcgtgt ttgccgttcc tgcgttagct gcgctggtag 4200
cgggcaataa cagcctgtgg gcgtggcccg ttttgattat cttagtgttc ccgattgcga 4260
ttgtgtttgc gattctgggt cgccactatc ccagcgcagg cggcgtcgcg cacttcgtcg 4320
gtatggcgtt tggttcgcgg cttgagcgag tcaccggctg gctgttttta tcggtcattc 4380
ccgtgggttt gcctgccgca ctacaaattg ccgccgggtt cggccaggcg atgtttggct 4440
ggcatagctg gcaactgttg ttggcagaac tcggtacgct ggcgctggtg tggtatatcg 4500
gtactcgcgg tgccagttcc agtgctaatc tacaaaccgt tattgccgga cttatcgtcg 4560
cgctgattgt cgctatctgg tgggcgggcg atatcaaacc tgcgaatatc ccctttccgg 4620
cacctggtaa tatcgaactt accgggttat ttgctgcgtt atcagtgatg ttctggtgtt 4680
ttgtcggtct ggaggcattt gcccatctcg cctcggaatt taaaaatcca gagcgtgatt 4740
ttcctcgtgc tttgatgatt ggtctgctgc tggcaggatt agtctactgg ggctgtacgg 4800
tagtcgtctt acacttcgac gcctatggtg aaaaaatggc ggcggcagca tcgcttccaa 4860
aaattgtagt gcagttgttc ggtgtaggag cgttatggat tgcctgcgtg attggctatc 4920
tggcctgctt tgccagtctc aacatttata tacagagctt cgcccgcctg gtctggtcgc 4980
aggcgcaaca taatcctgac cactacctgg cacgcctctc ttctcgccat atcccgaata 5040
atgccctcaa tgcggtgctc ggctgctgtg tggtgagcac tttggtgatt catgctttag 5100
agatcaatct ggacgctctt attatttatg ccaatggcat ctttattatg atttatctgt 5160
tatgcatgct ggcaggctgt aaattattgc aaggacgtta tcgactactg gcggtggttg 5220
gcgggctgtt atgcgttctg ttactggcaa tggtcggctg gaaaagtctc tatgcgctga 5280
tcatgctggc ggggttatgg ctgttgctgc caaaacgaaa aacgccggaa aatggcataa 5340
ccacataacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 5400
gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt 5460
caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc 5520
gacgaccggg tcatcgtggc cggatcttgc ggcccctcgg cttgaacgaa ttgttagaca 5580
ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg 5640
atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag 5700
tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt 5760
cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt 5820
tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc 5880
ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa 5940
ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc 6000
tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg 6060
cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc 6120
gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc 6180
tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg 6240
tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc 6300
gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac 6360
cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 6420
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagcta gctcactcgg 6480
tcgctacgct ccgggcgtga gactgcggcg g 6511
<210> 4
<211> 8632
<212> DNA
<213> Synthesis
<400> 4
gcgctgcgga cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg 60
tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca 120
gagttcacat aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg 180
aatctgacag tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt 240
cgctccctct tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt 300
tctcccttac gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt 360
gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg 420
cgccttatcc ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact 480
ggcagcagcc attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg 540
ctcttgaagt gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc 600
tgcgaaagcc agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc 660
acctccccag gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc 720
tcaagaagat cctttgatct tttctactga gctgattttc tgcgcccaac attgttggga 780
ttgatgggcc gtagatatcg gcgtccagaa tgccgacttt cgccccttct gccgccagcg 840
ccagtgccag gtttaccgcc gtggacgatt tacccacgcc gcctttaccg gaactcaccg 900
cgataatgtt cttcacaccg ttaattcccg gctgattctt cacgcgtttc agagtggcta 960
tgttatgcga cagtttccag tcgatagctt tcgccccggt gatgcgcagc agttcggcgc 1020
tacattgctc ttttaactct tcgaacgcgc tatgccagac aaacggcata accagttcca 1080
catgcaacgt gtcgtccatc caggcgacat ggtgcaacgc tttcagcgtg gtgaggttat 1140
gcttcagggt tgggtgctga aaattggcca gtgtcccggc gaccattgcg cgcaaggctt 1200
ctggtgattt ggcctgggat tgttcgttca tcccgactcc ttttttgtat agataaacca 1260
tcagctgata gtttacctga agaatataga gaagtactta cttaacattt tcccatttgg 1320
tactatctaa ccccttttca ctattaagaa gtaatgccta ctatgactca agtcgcgaag 1380
aaaattctgg tgacgtgcgc actgccgtac gctaacggct caatccacct cggccatatg 1440
ctggagcaca tccaggctga tgtctgggtc cgttaccagc gaatgcgcgg ccacgaggtc 1500
aacttcatct gcgccgacga tgcccacggt acaccgatca tgctgaaagc tcagcagctt 1560
ggtatcaccc cggagcagat gattggcgaa atgagtcagg agcatcagac tgatttcgca 1620
ggctttaaca tcagctatga caactatcac tcgacgcaca gcgaagagaa ccgccagttg 1680
tcagaactta tctactctcg cctgaaagaa aacggtttta ttaaaaaccg caccatctct 1740
cagctgtacg atccggaaaa aggcatgttc ctgccggacc gttttgtgaa aggcacctgc 1800
ccgaaatgta aatccccgga tcaatacggc gataactgcg aagtctgcgg cgcgacctac 1860
agcccgactg aactgatcga gccgaaatcg gtggtttctg gcgctacgcc ggtaatgcgt 1920
gattctgaac acttcttctt tgatctgccc tctttcagcg aaatgttgca ggcatggacc 1980
cgcagcggtg cgttgcagga gcaggtggca aataaaatgc aggagtggtt tgaatctggc 2040
ctgcaacagt gggatatctc ccgcgacgcc ccttacttcg gttttgaaat tccgaacgcg 2100
ccgggcaaat atttctacgt ctggctggac gcaccgattg gctacatggg ttctttcaag 2160
aatctgtgcg acaagcgcgg cgacagcgta agcttcgatg aatactggaa gaaagactcc 2220
accgccgagc tgtaccactt catcggtaaa gatattgttt acttccacag cctgttctgg 2280
cctgccatgc tggaaggcag caacttccgc aagccgtcca acctgtttgt tcatggctat 2340
gtgacggtga acggcgcaaa gatgtccaag tctcgcggca cctttattaa agccagcacc 2400
tggctgaatc attttgacgc agacagcctg cgttactact acactgcgaa actctcttcg 2460
cgcattgatg atatcgatct caacctggaa gatttcgttc agcgtgtgaa tgccgatatc 2520
gttaacaaag tggttaacct ggcctcccgt aatgcgggct ttatcaacaa gcgttttgac 2580
ggcgtgctgg caagcgaact ggctgacccg cagttgtaca aaaccttcac tgatgccgct 2640
gaagtgattg gtgaagcgtg ggaaagccgt gaatttggta aagccgtgcg cgaaatcatg 2700
gcgctggctg atctggctaa ccgctatgtc gatgaacaag ctccgtgggt ggtggcgaaa 2760
caggaaggcc gcgatgccga cctgcaggca atttgctcaa tgggcatcaa cctgttccgc 2820
gtgctgatga cttacctgaa gccggtactg ccgaaactga ccgagcgtgc agaagcattc 2880
ctcaatacgg aactgacctg ggatggtatc cagcaaccgc tgctgggcca caaagtgaat 2940
ccgttcaagg cgctgtataa ccgcatcgat atgaggcagg ttgaagcact ggtggaagcc 3000
tctaaagaat aacatggact cgtctactag cgcagctgag cgcaacgcaa ttaatgtgag 3060
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 3120
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt 3180
cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 3240
gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 3300
gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac 3360
cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg 3420
tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc 3480
ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca 3540
catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg 3600
ttaactcggc gtttcatctg tggtgcaacg ggcgctgggt cggttacggc caggacagtc 3660
gtttgccgtc tgaatttgac ctgagcgcat ttttacgcgc cggagaaaac cgcctcgcgg 3720
tgatggtgct gcgctggagt gacggcagtt atctggaaga tcaggatatg tggcggatga 3780
gcggcatttt ccgtgacgtc tcgttgctgc ataaaccgac tacacaaatc agcgatttcc 3840
atgttgccac tcgctttaat gatgatttca gccgcgctgt actggaggct gaagttcaga 3900
tgtgcggcga gttgcgtgac tacctacggg taacagtttc tttatggcag ggtgaaacgc 3960
aggtcgccag cggcaccgcg cctttcggcg gtgaaattat cgatgagcgt ggtggttatg 4020
ccgatcgcgt cacactacgt ctgaacgtcg aaaacccgaa actgtggagc gccgaaatcc 4080
cgaatctcta tcgtgcggtg gttgaactgc acaccgccga cggcacgctg attgaagcag 4140
aagcctgcga tgtcggtttc cgcgaggtgc ggattgaaaa tggtctgctg ctgctgaacg 4200
gcaagccgtt gctgattcga ggcgttaacc gtcacgagca tcatcctctg catggtcagg 4260
tcatggatga gcagacgatg gtgcaggata tcctgctgat gaagcagaac aactttaacg 4320
ccgtgcgctg ttcgcattat ccgaaccatc cgctgtggta cacgctgtgc gaccgctacg 4380
gcctgtatgt ggtggatgaa gccaatattg aaacccacgg catggtgcca atgaatcgtc 4440
tgaccgatga tccgcgctgg ctaccggcga tgagcgaacg cgtaacgcga atggtgcagc 4500
gcgatcgtaa tcacccgagt gtgatcatct ggtcgctggg gaatgaatca ggccacggcg 4560
ctaatcacga cgcgctgtat cgctggatca aatctgtcga tccttcccgc ccggtgcagt 4620
atgaaggcgg cggagccgac accacggcca ccgatattat ttgcccgatg tacgcgcgcg 4680
tggatgaaga ccagcccttc ccggctgtgc cgaaatggtc catcaaaaaa tggctttcgc 4740
tacctggaga gacgcgcccg ctgatccttt gcgaatacgc ccacgcgatg ggtaacagtc 4800
ttggcggttt cgctaaatac tggcaggcgt ttcgtcagta tccccgttta cagggcggct 4860
tcgtctggga ctgggtggat cagtcgctga ttaaatatga tgaaaacggc aacccgtggt 4920
cggcttacgg cggtgatttt ggcgatacgc cgaacgatcg ccagttctgt atgaacggtc 4980
tggtctttgc cgaccgcacg ccgcatccag cgctgacgga agcaaaacac cagcagcagt 5040
ttttccagtt ccgtttatcc gggcaaacca tcgaagtgac cagcgaatac ctgttccgtc 5100
atagcgataa cgagctcctg cactggatgg tggcgctgga tggtaagccg ctggcaagcg 5160
gtgaagtgcc tctggatgtc gctccacaag gtaaacagtt gattgaactg cctgaactac 5220
cgcagccgga gagcgccggg caactctggc tcacagtacg cgtagtgcaa ccgaacgcga 5280
ccgcatggtc agaagccggg cacatcagcg cctggcagca gtggcgtctg gcggaaaacc 5340
tcagtgtgac gctccccgcc gcgtcccacg ccatcccgca tctgaccacc agcgaaatgg 5400
atttttgcat cgagctgggt aataagcgtt ggcaatttaa ccgccagtca ggctttcttt 5460
cacagatgtg gattggcgat aaaaaacaac tgctgacgcc gctgcgcgat cagttcaccc 5520
gtgcaccgct ggataacgac attggcgtaa gtgaagcgac ccgcattgac cctaacgcct 5580
gggtcgaacg ctggaaggcg gcgggccatt accaggccga agcagcgttg ttgcagtgca 5640
cggcagatac acttgctgat gcggtgctga ttacgaccgc tcacgcgtgg cagcatcagg 5700
ggaaaacctt atttatcagc cggaaaacct accggattga tggtagtggt caaatggcga 5760
ttaccgttga tgttgaagtg gcgagcgata caccgcatcc ggcgcggatt ggcctgaact 5820
gccagctggc gcaggtagca gagcgggtaa actggctcgg attagggccg caagaaaact 5880
atcccgaccg ccttactgcc gcctgttttg accgctggga tctgccattg tcagacatgt 5940
ataccccgta cgtcttcccg agcgaaaacg gtctgcgctg cgggacgcgc gaattgaatt 6000
atggcccaca ccagtggcgc ggcgacttcc agttcaacat cagccgctac agtcaacagc 6060
aactgatgga aaccagccat cgccatctgc tgcacgcgga agaaggcaca tggctgaata 6120
tcgacggttt ccatatgggg attggtggcg acgactcctg gagcccgtca gtatcggcgg 6180
aattccagct gagcgccggt cgctaccatt accagttggt ctggtgtcaa aaataattaa 6240
ttaaggaatt gtgagcggat aacaatttca cacaggaaac agacccctag gctgctgcca 6300
ttgacaatta atcatccggc tcgtataatg tgtcacacag gaaacagacc atgagtggac 6360
tcaaacaaga actggggctg gcccagggca ttggcctgct atcgacgtca ttattaggca 6420
ctggcgtgtt tgccgttcct gcgttagctg cgctggtagc gggcaataac agcctgtggg 6480
cgtggcccgt tttgattatc ttagtgttcc cgattgcgat tgtgtttgcg attctgggtc 6540
gccactatcc cagcgcaggc ggcgtcgcgc acttcgtcgg tatggcgttt ggttcgcggc 6600
ttgagcgagt caccggctgg ctgtttttat cggtcattcc cgtgggtttg cctgccgcac 6660
tacaaattgc cgccgggttc ggccaggcga tgtttggctg gcatagctgg caactgttgt 6720
tggcagaact cggtacgctg gcgctggtgt ggtatatcgg tactcgcggt gccagttcca 6780
gtgctaatct acaaaccgtt attgccggac ttatcgtcgc gctgattgtc gctatctggt 6840
gggcgggcga tatcaaacct gcgaatatcc cctttccggc acctggtaat atcgaactta 6900
ccgggttatt tgctgcgtta tcagtgatgt tctggtgttt tgtcggtctg gaggcatttg 6960
cccatctcgc ctcggaattt aaaaatccag agcgtgattt tcctcgtgct ttgatgattg 7020
gtctgctgct ggcaggatta gtctactggg gctgtacggt agtcgtctta cacttcgacg 7080
cctatggtga aaaaatggcg gcggcagcat cgcttccaaa aattgtagtg cagttgttcg 7140
gtgtaggagc gttatggatt gcctgcgtga ttggctatct ggcctgcttt gccagtctca 7200
acatttatat acagagcttc gcccgcctgg tctggtcgca ggcgcaacat aatcctgacc 7260
actacctggc acgcctctct tctcgccata tcccgaataa tgccctcaat gcggtgctcg 7320
gctgctgtgt ggtgagcact ttggtgattc atgctttaga gatcaatctg gacgctctta 7380
ttatttatgc caatggcatc tttattatga tttatctgtt atgcatgctg gcaggctgta 7440
aattattgca aggacgttat cgactactgg cggtggttgg cgggctgtta tgcgttctgt 7500
tactggcaat ggtcggctgg aaaagtctct atgcgctgat catgctggcg gggttatggc 7560
tgttgctgcc aaaacgaaaa acgccggaaa atggcataac cacataaccg ctgagcaata 7620
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca 7680
ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca 7740
atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt catcgtggcc 7800
ggatcttgcg gcccctcggc ttgaacgaat tgttagacat tatttgccga ctaccttggt 7860
gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg 7920
atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc 7980
cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac 8040
tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc 8100
gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg 8160
aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc 8220
ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag 8280
aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg 8340
aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc 8400
aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag 8460
ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac 8520
tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc 8580
aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca ta 8632
<210> 5
<211> 9049
<212> DNA
<213> Synthesis
<400> 5
aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60
tctcgctaac ccaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120
aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180
attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240
atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttgga 300
cgcgtacaac tcaagtctga cataaatgac cgctatgagc actgcaatta cacgccagat 360
cgttctcgct accgcaacca ccggtatgaa ccagattggt gcgcactatg aaggccacaa 420
gatcattgag attggtgccg ttgaagtggt gaaccgtcgc ctgacgggca ataacttcca 480
tgtttatctc aaacccgatc ggctggtgga tccggaagcc tttggcgtac atggtattgc 540
cgatgaattt ttgctcgata agcccacgtt tgccgaagta gccgatgagt tcatggacta 600
tattcgcggc gcggagttgg tgatccataa cgcagcgttc gatatcggct ttatggacta 660
cgagttttcg ttgcttaagc gcgatattcc gaagaccaat actttctgta aggtcaccga 720
tagccttgcg gtggcgagga aaatgtttcc cggtaagcgc aacagcctcg atgcgttatg 780
tgctcgctac gaaatagata acagtaaacg aacgctgcac ggggcattac tcgatgccca 840
gatccttgcg gaagtttatc tggcgatgac cggtggtcaa acgtcgatgg cttttgcgat 900
ggaaggagag acacaacagc aacaaggtga agcaacaatt cagcgcattg tacgtcaggc 960
aagtaagtta cgcgttgttt ttgcgacaga tgaagagatt gcagctcatg aagcccgtct 1020
cgatctggtg cagaagaaag gcggaagttg cctctggcga gcataattta atatcagtaa 1080
accggacata acccatgaag aaaaatcgcg cttttttgaa gtgggcaggg ggcaagtatc 1140
ccctgcttga tgatattaaa cggcatttgc ccaagggcga atgtctggtt gagccttttg 1200
taggtgccgg gtcggtgttt ctcaacaccg acttttctcg ttatatcctt gccgatatca 1260
atagcgacct gatcagtctc tataacattg tgaagatgcg tactgatgag tacgtacagg 1320
ccgcacgcga gctgtttgtt cccgaaacaa attgcgccga ggtttactat cagttccgcg 1380
aagagttcaa caaaagccag gatccgttcc gtcgggcggt actgttttta tatttgaacc 1440
gctacggtta caacggcctg tgtcgttaca atctgcgcgg tgagtttaac gtgccgttcg 1500
gccgctacaa aaaaccctat ttcccggaag cagagttgta tcacttcgct gaaaaagcgc 1560
agaatgcctt tttctattgt gagtcttacg ccgatagcat ggcgcgcgca gatgatgcat 1620
ccgtcgtcta ttgcgatccg ccttatgcac cgctgtctgc gaccgccaac tttacggcgt 1680
atcacacaaa cagttttacg cttgaacaac aagcgcatct ggcggagatc gccgaaggtc 1740
tggttgagcg ccatattcca gtgctgatct ccaatcacga tacgatgtta acgcgtgagt 1800
ggtatcagcg cgcaaaattg catgtcgtca aagttcgacg cagtataagc agcaacggcg 1860
gcacacgtaa aaaggtggac gaactgctgg ctttgtacaa accaggagtc gtttcacccg 1920
cgaaaaaata attcagctaa gacactgcac tggattaaga tgaaaacgat tgaagttgat 1980
gatgaactct acagctatat tgccagccac actaagcata tcggcgagag cgcatccgac 2040
attttacggc gtatgttgaa attttccgcc gcatcacagc ctgctgctcc ggtgacgaaa 2100
gaggttcgcg ttgcgtcacc tgctatcgtc gaagcgaagc cggtcaaaac gattaaagac 2160
aaggttcgcg caatgcgtga acttctgctt tcggatgaat acgcagagca aaagcgagcg 2220
gtcaatcgct ttatgctgct gttgtctaca ctatattctc ttgacgccca ggcgtttgcc 2280
gaagcaacgg aatcgttgca cggtcgtaca cgcgtttact ttgcggcaga tgaacaaacg 2340
ctgctgaaaa atggtaatca gaccaagccg aaacatgtgc caggcacgcc gtattgggtg 2400
atcaccaaca ccaacaccgg ccgtaaatgc agcatgatcg aacacatcat gcagtcgatg 2460
caattcccgg cggaattgat tgagaaggtt tgcggaacta tctaacggct gaaattaatg 2520
aggtcatacc caaatggata gttcgtttac gcccattgaa caaatgctaa aatttcgcgc 2580
cagccgccac gaagattttc cttatcagga gatccttctg actcgtcttt gcatgcacat 2640
gcaaagcaag ctgctggaga accgcaataa aatgctgaag gctcagggaa ttaacgagac 2700
gttgtttatg gcgttgatta cgctggagtc tcaggaaaac cacagtattc agccttctga 2760
attaagttgt gctcttggat catcccgtac caacgcgacg cgtattgccg atgaactgga 2820
aaaacgcggt tggatcgaac gtcgtgaaag cgataacgat cgccgctgcc tgcatctgca 2880
attaacggaa aaaggtcacg agtttttgcg cgaggtttta ccaccgcagc ataactgcct 2940
gcatcaactc tggtccgcgc tcagcacaac agaaaaagat cagctcgagc aaatcacccg 3000
caaattgctc tcccgtctcg accagatgga acaagacggt gtggttctcg aagcgatgag 3060
ctaataatac aaaaattagg aggaatttca acatgacaaa tttatctgac atcattgaaa 3120
aagaaacagg aaaacaacta gtgattcaag aatcaattct aatgttacca gaagaagtag 3180
aggaagtaat tgggaataaa ccagaaagtg atattttagt tcatactgct tatgatgaaa 3240
gtacagatga aaatgtaatg ctattaactt cagatgctcc agaatataaa ccttgggctt 3300
tagtaattca agacagtaat ggagaaaata aaattaaaat gttataagtc gagattaagt 3360
aaaccggaat ctgaagatga ccgacgcgga atacgttcgt atccacgaaa aactggacat 3420
ctacaccttc aaaaaacagt tcttcaacaa caaaaaatct gtttctcacc gttgctacgt 3480
tctgttcgaa ctgaaacgtc gtggtgaacg tcgtgcgtgc ttctggggtt acgcggttaa 3540
caaaccgcag tctggtaccg aacgtggtat ccacgcggaa atcttctcta tccgtaaagt 3600
tgaagaatac ctgcgtgaca acccgggtca gttcaccatc aactggtact cttcttggtc 3660
tccgtgcgcg gactgcgcgg aaaaaatcct ggaatggtac aaccaggaac tgcgtggtaa 3720
cggtcacacc ctgaaaatct gggcgtgcaa actgtactac gaaaaaaacg cgcgtaacca 3780
gatcggtctg tggaacctgc gtgacaacgg tgttggtctg aacgttatgg tttctgaaca 3840
ctaccagtgc tgccgtaaaa tcttcatcca gtcttctcac aaccagctga acgaaaaccg 3900
ttggctggaa aaaaccctga aacgtgcgga aaaacgtcgt tctgaactgt ctatcatgat 3960
ccaggttaaa atcctgcaca ccaccaaatc tccggcggtt taaacttaat taacggcact 4020
cctcagccag aagttcctat tctctagaaa gtataggaac ttcagtcaaa agcctccggt 4080
cggaggcttt tgactacatg cccatggcgt ttacgccccg ccctgccact catcgcagta 4140
ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac 4200
ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa 4260
aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 4320
ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 4380
gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 4440
gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 4500
agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg 4560
atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 4620
tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 4680
ttgagtaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 4740
ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga 4800
taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct 4860
tacgtgccaa gccaaatagg ccgtcactcg gtcgctacgc tccgggcgtg agactgcggc 4920
gggaagttcc tattctctag aaagtatagg aacttcgtga ttttgatgac gagcgtaatg 4980
gctggcctgt tgaacaagtc tggaaagaaa tgcagattct tgctcaattg ttatcagcta 5040
tgcgccgacc agaacacctt gccgatcagc caaacgtctc ttcaggccac tgactagcga 5100
taactttccc cacaacggaa caactctcat tgcatgggat cattgggtac tgtgggttta 5160
gtggttgtaa aaacacctga ccgctatccc tgatcagttt cttgaaggta aactcatcac 5220
ccccaagtct ggctatgcag aaatcacctg gctcaacagc ctgctcaggg tcaacgagaa 5280
ttaacattcc gtcaggaaag cttggcttgg agcctgttgg tgcggtcatg gaattacctt 5340
caacctcaag ccagaatgca gaatcactgg cttttttggt tgtgcttacc catctctccg 5400
catcaccttt ggtaaaggtt ctaagcttag gtgagaacat ccctgcctga acatgagaaa 5460
aaacagggta ctcatactca cttctaagtg acggctgcat actaaccgct tcatacatct 5520
cgtagatttc tctggcgatt gaagggctaa attcttcaac gctaactttg agaatttttg 5580
taagcaatgc ggcgttataa gcatttaatg cattgatgcc attaaataaa gcaccaacgc 5640
ctgactgccc catccccatc ttgtctgcga cagattcctg ggataagcca agttcatttt 5700
tctttttttc ataaattgct ttaaggcgac gtgcgtcctc aagctgctct tgtgttaatg 5760
gtttcttttt tgtgctcata cgttaaatct atcaccgcaa gggataaata tctaacaccg 5820
tgcgtgttga ctattttacc tctggcggtg ataatggttg catgtactaa ggaggttgta 5880
tgccacaatt tgatatatta tgtaaaacac cacctaaggt gcttgttcgt cagtttgtgg 5940
aaaggtttga aagaccttca ggtgagaaaa tagcattatg tgctgctgaa ctaacctatt 6000
tatgttggat gattacacat aacggaacag caatcaagag agccacattc atgagctata 6060
atactatcat aagcaattcg ctgagtttcg atattgtcaa taaatcactc cagtttaaat 6120
acaagacgca aaaagcaaca attctggaag cctcattaaa gaaattgatt cctgcttggg 6180
aatttacaat tattccttac tatggacaaa aacatcaatc tgatatcact gatattgtaa 6240
gtagtttgca attacagttc gaatcatcgg aagaagcaga taagggaaat agccacagta 6300
aaaaaatgct taaagcactt ctaagtgagg gtgaaagcat ctgggagatc actgagaaaa 6360
tactaaattc gtttgagtat acttcgagat ttacaaaaac aaaaacttta taccaattcc 6420
tcttcctagc tactttcatc aattgtggaa gattcagcga tattaagaac gttgatccga 6480
aatcatttaa attagtccaa aataagtatc tgggagtaat aatccagtgt ttagtgacag 6540
agacaaagac aagcgttagt aggcacatat acttctttag cgcaaggggt aggatcgatc 6600
cacttgtata tttggatgaa tttttgagga attctgaacc agtcctaaaa cgagtaaata 6660
ggaccggcaa ttcttcaagc aataaacagg aataccaatt attaaaagat aacttagtca 6720
gatcgtacaa taaagctttg aagaaaaatg cgccttattc aatctttgct ataaaaaatg 6780
gcccaaaatc tcacattgga agacatttga tgacctcatt tctttcaatg aagggcctaa 6840
cggagttgac taatgttgtg ggaaattgga gcgataagcg tgcttctgcc gtggccagga 6900
caacgtatac tcatcagata acagcaatac ctgatcacta cttcgcacta gtttctcggt 6960
actatgcata tgatccaata tcaaaggaaa tgatagcatt gaaggatgag actaatccaa 7020
ttgaggagtg gcagcatata gaacagctaa agggtagtgc tgaaggaagc atacgatacc 7080
ccgcatggaa tgggataata tcacaggagg tactagacta cctttcatcc tacataaata 7140
gacgcatata agtacgcatt taagcataaa cacgcactat gccgttcttc tcatgtatat 7200
atatatacag gcaacacgca gatataggtg cgacgtgaac agtgagctgt atgtgcgcgc 7260
tgcggacaca tacaaagtta cccacagatt ccgtggataa gcaggggact aacatgtgag 7320
gcaaaacagc agggccgcgc cggtggcgtt tttccatagg ctccgccctc ctgccagagt 7380
tcacataaac agacgctttt ccggtgcatc tgtgggagcc gtgaggctca accatgaatc 7440
tgacagtacg ggcgaaaccc gacaggactt aaagatcccc accgtttccg gcgggtcgct 7500
ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg atacctgttc cgcctttctc 7560
ccttacggga agtgtggcgc tttctcatag ctcacacact ggtatctcgg ctcggtgtag 7620
gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg ttcagcccga ctgctgcgcc 7680
ttatccggta actgttcact tgagtccaac ccggaaaagc acggtaaaac gccactggca 7740
gcagccattg gtaactggga gttcgcagag gatttgttta gctaaacacg cggttgctct 7800
tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga tttggttgct gtgctctgcg 7860
aaagccagtt accacggtta agcagttccc caactgactt aaccttcgat caaaccacct 7920
ccccaggtgg ttttttcgtt tacagggcaa aagattacgc gcagaaaaaa aggatctcaa 7980
gaagatcctt tgatcttttc tactgaaccg ctctagattt cagtgcaatt tatctcttca 8040
aatgtagcac ctgaagtcag cccaggagga agaggacatc cggtcaaata aaacgaaagg 8100
ctcagtcgaa agactgggcc tttcgtttta gacttaggga ccctttatga caacttgacg 8160
gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 8220
cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 8280
gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 8340
tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 8400
ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 8460
tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 8520
ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 8580
gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 8640
ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 8700
agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 8760
gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 8820
ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 8880
ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 8940
gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 9000
ggatcatttt gcgcttcagc catacttttc atactcccac cattcagag 9049
<210> 6
<211> 11310
<212> DNA
<213> Synthesis
<400> 6
aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60
tctcgctaac ccaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120
aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180
attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240
atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttgga 300
cgcgtacaac tcaagtctga cataaatgac cgctatgagc actgcaatta cacgccagat 360
cgttctcgct accgcaacca ccggtatgaa ccagattggt gcgcactatg aaggccacaa 420
gatcattgag attggtgccg ttgaagtggt gaaccgtcgc ctgacgggca ataacttcca 480
tgtttatctc aaacccgatc ggctggtgga tccggaagcc tttggcgtac atggtattgc 540
cgatgaattt ttgctcgata agcccacgtt tgccgaagta gccgatgagt tcatggacta 600
tattcgcggc gcggagttgg tgatccataa cgcagcgttc gatatcggct ttatggacta 660
cgagttttcg ttgcttaagc gcgatattcc gaagaccaat actttctgta aggtcaccga 720
tagccttgcg gtggcgagga aaatgtttcc cggtaagcgc aacagcctcg atgcgttatg 780
tgctcgctac gaaatagata acagtaaacg aacgctgcac ggggcattac tcgatgccca 840
gatccttgcg gaagtttatc tggcgatgac cggtggtcaa acgtcgatgg cttttgcgat 900
ggaaggagag acacaacagc aacaaggtga agcaacaatt cagcgcattg tacgtcaggc 960
aagtaagtta cgcgttgttt ttgcgacaga tgaagagatt gcagctcatg aagcccgtct 1020
cgatctggtg cagaagaaag gcggaagttg cctctggcga gcataattta atatcagtaa 1080
accggacata acccatgaag aaaaatcgcg cttttttgaa gtgggcaggg ggcaagtatc 1140
ccctgcttga tgatattaaa cggcatttgc ccaagggcga atgtctggtt gagccttttg 1200
taggtgccgg gtcggtgttt ctcaacaccg acttttctcg ttatatcctt gccgatatca 1260
atagcgacct gatcagtctc tataacattg tgaagatgcg tactgatgag tacgtacagg 1320
ccgcacgcga gctgtttgtt cccgaaacaa attgcgccga ggtttactat cagttccgcg 1380
aagagttcaa caaaagccag gatccgttcc gtcgggcggt actgttttta tatttgaacc 1440
gctacggtta caacggcctg tgtcgttaca atctgcgcgg tgagtttaac gtgccgttcg 1500
gccgctacaa aaaaccctat ttcccggaag cagagttgta tcacttcgct gaaaaagcgc 1560
agaatgcctt tttctattgt gagtcttacg ccgatagcat ggcgcgcgca gatgatgcat 1620
ccgtcgtcta ttgcgatccg ccttatgcac cgctgtctgc gaccgccaac tttacggcgt 1680
atcacacaaa cagttttacg cttgaacaac aagcgcatct ggcggagatc gccgaaggtc 1740
tggttgagcg ccatattcca gtgctgatct ccaatcacga tacgatgtta acgcgtgagt 1800
ggtatcagcg cgcaaaattg catgtcgtca aagttcgacg cagtataagc agcaacggcg 1860
gcacacgtaa aaaggtggac gaactgctgg ctttgtacaa accaggagtc gtttcacccg 1920
cgaaaaaata attcagctaa gacactgcac tggattaaga tgaaaacgat tgaagttgat 1980
gatgaactct acagctatat tgccagccac actaagcata tcggcgagag cgcatccgac 2040
attttacggc gtatgttgaa attttccgcc gcatcacagc ctgctgctcc ggtgacgaaa 2100
gaggttcgcg ttgcgtcacc tgctatcgtc gaagcgaagc cggtcaaaac gattaaagac 2160
aaggttcgcg caatgcgtga acttctgctt tcggatgaat acgcagagca aaagcgagcg 2220
gtcaatcgct ttatgctgct gttgtctaca ctatattctc ttgacgccca ggcgtttgcc 2280
gaagcaacgg aatcgttgca cggtcgtaca cgcgtttact ttgcggcaga tgaacaaacg 2340
ctgctgaaaa atggtaatca gaccaagccg aaacatgtgc caggcacgcc gtattgggtg 2400
atcaccaaca ccaacaccgg ccgtaaatgc agcatgatcg aacacatcat gcagtcgatg 2460
caattcccgg cggaattgat tgagaaggtt tgcggaacta tctaacggct gaaattaatg 2520
aggtcatacc caaatggata gttcgtttac gcccattgaa caaatgctaa aatttcgcgc 2580
cagccgccac gaagattttc cttatcagga gatccttctg actcgtcttt gcatgcacat 2640
gcaaagcaag ctgctggaga accgcaataa aatgctgaag gctcagggaa ttaacgagac 2700
gttgtttatg gcgttgatta cgctggagtc tcaggaaaac cacagtattc agccttctga 2760
attaagttgt gctcttggat catcccgtac caacgcgacg cgtattgccg atgaactgga 2820
aaaacgcggt tggatcgaac gtcgtgaaag cgataacgat cgccgctgcc tgcatctgca 2880
attaacggaa aaaggtcacg agtttttgcg cgaggtttta ccaccgcagc ataactgcct 2940
gcatcaactc tggtccgcgc tcagcacaac agaaaaagat cagctcgagc aaatcacccg 3000
caaattgctc tcccgtctcg accagatgga acaagacggt gtggttctcg aagcgatgag 3060
ctaataatac aaaaattagg aggaatttca acatgacaaa tttatctgac atcattgaaa 3120
aagaaacagg aaaacaacta gtgattcaag aatcaattct aatgttacca gaagaagtag 3180
aggaagtaat tgggaataaa ccagaaagtg atattttagt tcatactgct tatgatgaaa 3240
gtacagatga aaatgtaatg ctattaactt cagatgctcc agaatataaa ccttgggctt 3300
tagtaattca agacagtaat ggagaaaata aaattaaaat gttataagtc gagattaagt 3360
aaaccggaat ctgaagatga ccgacgcgga atacgttcgt atccacgaaa aactggacat 3420
ctacaccttc aaaaaacagt tcttcaacaa caaaaaatct gtttctcacc gttgctacgt 3480
tctgttcgaa ctgaaacgtc gtggtgaacg tcgtgcgtgc ttctggggtt acgcggttaa 3540
caaaccgcag tctggtaccg aacgtggtat ccacgcggaa atcttctcta tccgtaaagt 3600
tgaagaatac ctgcgtgaca acccgggtca gttcaccatc aactggtact cttcttggtc 3660
tccgtgcgcg gactgcgcgg aaaaaatcct ggaatggtac aaccaggaac tgcgtggtaa 3720
cggtcacacc ctgaaaatct gggcgtgcaa actgtactac gaaaaaaacg cgcgtaacca 3780
gatcggtctg tggaacctgc gtgacaacgg tgttggtctg aacgttatgg tttctgaaca 3840
ctaccagtgc tgccgtaaaa tcttcatcca gtcttctcac aaccagctga acgaaaaccg 3900
ttggctggaa aaaaccctga aacgtgcgga aaaacgtcgt tctgaactgt ctatcatgat 3960
ccaggttaaa atcctgcaca ccaccaaatc tccggcggtt taaacttaat taacggcact 4020
cctcagccag aagttcctat tctctagaaa gtataggaac ttcagtcaaa agcctccggt 4080
cggaggcttt tgactacatg cccatggcgt ttacgccccg ccctgccact catcgcagta 4140
ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac 4200
ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa 4260
aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 4320
ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 4380
gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 4440
gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 4500
agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg 4560
atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 4620
tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 4680
ttgagtaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 4740
ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga 4800
taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct 4860
tacgtgccaa gccaaatagg ccgtcactcg gtcgctacgc tccgggcgtg agactgcggc 4920
ggttattctt tagaggcttc caccagtgct tcaacctgcc tcatatcgat gcggttatac 4980
agcgccttga acggattcac tttgtggccc agcagcggtt gctggatacc atcccaggtc 5040
agttccgtat tgaggaatgc ttctgcacgc tcggtcagtt tcggcagtac cggcttcagg 5100
taagtcatca gcacgcggaa caggttgatg cccattgagc aaattgcctg caggtcggca 5160
tcgcggcctt cctgtttcgc caccacccac ggagcttgtt catcgacata gcggttagcc 5220
agatcagcca gcgccatgat ttcgcgcacg gctttaccaa attcacggct ttcccacgct 5280
tcaccaatca cttcagcggc atcagtgaag gttttgtaca actgcgggtc agccagttcg 5340
cttgccagca cgccgtcaaa acgcttgttg ataaagcccg cattacggga ggccaggtta 5400
accactttgt taacgatatc ggcattcaca cgctgaacga aatcttccag gttgagatcg 5460
atatcatcaa tgcgcgaaga gagtttcgca gtgtagtagt aacgcaggct gtctgcgtca 5520
aaatgattca gccaggtgct ggctttaata aaggtgccgc gagacttgga catctttgcg 5580
ccgttcaccg tcacatagcc atgaacaaac aggttggacg gcttgcggaa gttgctgcct 5640
tccagcatgg caggccagaa caggctgtgg aagtaaacaa tatctttacc gatgaagtgg 5700
tacagctcgg cggtggagtc tttcttccag tattcatcga agcttacgct gtcgccgcgc 5760
ttgtcgcaca gattcttgaa agaacccatg tagccaatcg gtgcgtccag ccagacgtag 5820
aaatatttgc ccggcgcgtt cggaatttca aaaccgaagt aaggggcgtc gcgggagata 5880
tcccactgtt gcaggccaga ttcaaaccac tcctgcattt tatttgccac ctgctcctgc 5940
aacgcaccgc tgcgggtcca tgcctgcaac atttcgctga aagagggcag atcaaagaag 6000
aagtgttcag aatcacgcat taccggcgta gcgccagaaa ccaccgattt cggctcgatc 6060
agttcagtcg ggctgtaggt cgcgccgcag acttcgcagt tatcgccgta ttgatccggg 6120
gatttacatt tcgggcaggt gcctttcaca aaacggtccg gcaggaacat gcctttttcc 6180
ggatcgtaca gctgagagat ggtgcggttt ttaataaaac cgttttcttt caggcgagag 6240
tagataagtt ctgacaactg gcggttctct tcgctgtgcg tcgagtgata gttgtcatag 6300
ctgatgttaa agcctgcgaa atcagtctga tgctcctgac tcatttcgcc aatcatctgc 6360
tccggggtga taccaagctg ctgagctttc agcatgatcg gtgtaccgtg ggcatcgtcg 6420
gcgcagatga agttgacctc gtggccgcgc attcgctggt aacggaccca gacatcagcc 6480
tggatgtgct ccagcatatg gccgaggtgg attgagccgt tagcgtacgg cagtgcgcac 6540
gtcaccagaa ttttcttcgc gacttgagtc atagtaggca ttacttctta atagtgaaaa 6600
gggttagata gtaccaaatg ggaaaatgtt aagtaagtac ttctctatat tcttcaggta 6660
aactatcagc tgatggttta tctatacaaa aaaggagtcg ggatgaacga acaatcccag 6720
gccaaatcac cagaagcctt gcgcgcaatg gtcgccggga cactggccaa ttttcagcac 6780
ccaaccctga agcataacct caccacgctg aaagcgttgc accatgtcgc ctggatggac 6840
gacacgttgc atgtggaact ggttatgccg tttgtctggc atagcgcgtt cgaagagtta 6900
aaagagcaat gtagcgccga actgctgcgc atcaccgggg cgaaagctat cgactggaaa 6960
ctgtcgcata acatagccac tctgaaacgc gtgaagaatc agccgggaat taacggtgtg 7020
aagaacatta tcgcggtgag ttccggtaaa ggcggcgtgg gtaaatcgtc cacggcggta 7080
aacctggcac tggcgctggc ggcagaaggg gcgaaagtcg gcattctgga cgccgatatc 7140
tacggcccat caatcccaac aatgttgggc gcagaaaatc agcgaagttc ctattctcta 7200
gaaagtatag gaacttcgtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 7260
ctggaaagaa atgcagattc ttgctcaatt gttatcagct atgcgccgac cagaacacct 7320
tgccgatcag ccaaacgtct cttcaggcca ctgactagcg ataactttcc ccacaacgga 7380
acaactctca ttgcatggga tcattgggta ctgtgggttt agtggttgta aaaacacctg 7440
accgctatcc ctgatcagtt tcttgaaggt aaactcatca cccccaagtc tggctatgca 7500
gaaatcacct ggctcaacag cctgctcagg gtcaacgaga attaacattc cgtcaggaaa 7560
gcttggcttg gagcctgttg gtgcggtcat ggaattacct tcaacctcaa gccagaatgc 7620
agaatcactg gcttttttgg ttgtgcttac ccatctctcc gcatcacctt tggtaaaggt 7680
tctaagctta ggtgagaaca tccctgcctg aacatgagaa aaaacagggt actcatactc 7740
acttctaagt gacggctgca tactaaccgc ttcatacatc tcgtagattt ctctggcgat 7800
tgaagggcta aattcttcaa cgctaacttt gagaattttt gtaagcaatg cggcgttata 7860
agcatttaat gcattgatgc cattaaataa agcaccaacg cctgactgcc ccatccccat 7920
cttgtctgcg acagattcct gggataagcc aagttcattt ttcttttttt cataaattgc 7980
tttaaggcga cgtgcgtcct caagctgctc ttgtgttaat ggtttctttt ttgtgctcat 8040
acgttaaatc tatcaccgca agggataaat atctaacacc gtgcgtgttg actattttac 8100
ctctggcggt gataatggtt gcatgtacta aggaggttgt atgccacaat ttgatatatt 8160
atgtaaaaca ccacctaagg tgcttgttcg tcagtttgtg gaaaggtttg aaagaccttc 8220
aggtgagaaa atagcattat gtgctgctga actaacctat ttatgttgga tgattacaca 8280
taacggaaca gcaatcaaga gagccacatt catgagctat aatactatca taagcaattc 8340
gctgagtttc gatattgtca ataaatcact ccagtttaaa tacaagacgc aaaaagcaac 8400
aattctggaa gcctcattaa agaaattgat tcctgcttgg gaatttacaa ttattcctta 8460
ctatggacaa aaacatcaat ctgatatcac tgatattgta agtagtttgc aattacagtt 8520
cgaatcatcg gaagaagcag ataagggaaa tagccacagt aaaaaaatgc ttaaagcact 8580
tctaagtgag ggtgaaagca tctgggagat cactgagaaa atactaaatt cgtttgagta 8640
tacttcgaga tttacaaaaa caaaaacttt ataccaattc ctcttcctag ctactttcat 8700
caattgtgga agattcagcg atattaagaa cgttgatccg aaatcattta aattagtcca 8760
aaataagtat ctgggagtaa taatccagtg tttagtgaca gagacaaaga caagcgttag 8820
taggcacata tacttcttta gcgcaagggg taggatcgat ccacttgtat atttggatga 8880
atttttgagg aattctgaac cagtcctaaa acgagtaaat aggaccggca attcttcaag 8940
caataaacag gaataccaat tattaaaaga taacttagtc agatcgtaca ataaagcttt 9000
gaagaaaaat gcgccttatt caatctttgc tataaaaaat ggcccaaaat ctcacattgg 9060
aagacatttg atgacctcat ttctttcaat gaagggccta acggagttga ctaatgttgt 9120
gggaaattgg agcgataagc gtgcttctgc cgtggccagg acaacgtata ctcatcagat 9180
aacagcaata cctgatcact acttcgcact agtttctcgg tactatgcat atgatccaat 9240
atcaaaggaa atgatagcat tgaaggatga gactaatcca attgaggagt ggcagcatat 9300
agaacagcta aagggtagtg ctgaaggaag catacgatac cccgcatgga atgggataat 9360
atcacaggag gtactagact acctttcatc ctacataaat agacgcatat aagtacgcat 9420
ttaagcataa acacgcacta tgccgttctt ctcatgtata tatatataca ggcaacacgc 9480
agatataggt gcgacgtgaa cagtgagctg tatgtgcgcg ctgcggacac atacaaagtt 9540
acccacagat tccgtggata agcaggggac taacatgtga ggcaaaacag cagggccgcg 9600
ccggtggcgt ttttccatag gctccgccct cctgccagag ttcacataaa cagacgcttt 9660
tccggtgcat ctgtgggagc cgtgaggctc aaccatgaat ctgacagtac gggcgaaacc 9720
cgacaggact taaagatccc caccgtttcc ggcgggtcgc tccctcttgc gctctcctgt 9780
tccgaccctg ccgtttaccg gatacctgtt ccgcctttct cccttacggg aagtgtggcg 9840
ctttctcata gctcacacac tggtatctcg gctcggtgta ggtcgttcgc tccaagctgg 9900
gctgtaagca agaactcccc gttcagcccg actgctgcgc cttatccggt aactgttcac 9960
ttgagtccaa cccggaaaag cacggtaaaa cgccactggc agcagccatt ggtaactggg 10020
agttcgcaga ggatttgttt agctaaacac gcggttgctc ttgaagtgtg cgccaaagtc 10080
cggctacact ggaaggacag atttggttgc tgtgctctgc gaaagccagt taccacggtt 10140
aagcagttcc ccaactgact taaccttcga tcaaaccacc tccccaggtg gttttttcgt 10200
ttacagggca aaagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 10260
ctactgaacc gctctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca 10320
gcccaggagg aagaggacat ccggtcaaat aaaacgaaag gctcagtcga aagactgggc 10380
ctttcgtttt agacttaggg accctttatg acaacttgac ggctacatca ttcacttttt 10440
cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 10500
agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 10560
tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 10620
taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 10680
gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 10740
cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 10800
gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 10860
gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 10920
ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 10980
cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 11040
agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 11100
gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 11160
ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 11220
ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 11280
ccatactttt catactccca ccattcagag 11310
<210> 7
<211> 5308
<212> DNA
<213> Synthesis
<400> 7
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 60
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 120
ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 180
atgctggatc cttgacagct agctcagtcc taggtataat actagtcggt gctaaaccgg 240
gtcatcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 300
aaaagtggca ccgagtcggt gctttttttg accttgacag ctagctcagt cctaggtata 360
atactagttc aagtcgcgaa gaaaattcgt tttagagcta gaaatagcaa gttaaaataa 420
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctggacg atttacccac 480
gccgccttta ccggaactca ccgcgataat gttcttcaca ccgttaattc ccggctgatt 540
cttcacgcgt ttcagagtgg ctatgttatg cgacagtttc cagtcgatag ctttcgcccc 600
ggtgatgcgc agcagttcgg cgctacattg ctcttttaac tcttcgaacg cgctatgcca 660
gacaaacggc ataaccagtt ccacatgcaa cgtgtcgtcc atccaggcga catggtgcaa 720
cgctttcagc gtggtgaggt tatgcttcag ggttgggtgc tgaaaattgg ccagtgtccc 780
ggcgaccatt gcgcgcaagg cttctggtga tttggcctgg gattgttcgt tcatcccgac 840
tccttttttg tatagataaa ccatcagctg atagtttacc tgaagaatat agagaagtac 900
ttacttaaca ttttcccatt tggtactatc taaccccttt tcactattaa gaagtaatgc 960
ctactatgta atcccccttc aaggcgctgc atcgacagcg ccttttcttt ataaattcct 1020
aaagttgttt tcttgcgatt ttgtctctct ctaacccgca taaatactgg tagcatctgc 1080
attcaactgg ataaaattac agggatgcag aatgagacac tttatctatc aggacgaaaa 1140
atcacataaa ttcagggcag ttgagcaaca gggaaacgag ttgcatatca gttggggaaa 1200
agttggcacc aaagggcaaa gccagataaa aagtttttca gatgctgcgg cagcggcaaa 1260
agcggagctt aagctgattg cggagaaggt gaagaagggg tatgtggagc aagcgaagga 1320
taattctttg caaccttccc aaacggtaac gggctctctc aaggtagcgg acttatccac 1380
cattattcag gaacaaccct ctttcgtagc agaaacccgt gcgcctgaca aaaatacaga 1440
tgctgtttta ccgtggctgg cgaaagatat tgtcgacctg cagaagctta gatctattac 1500
cctgttatcc ctactcgagt tcatgtgcag ctccataagc aaaaggggat gataagttta 1560
tcaccaccga ctatttgcaa cagtgccgtt gatcgtgcta tgatcgactg atgtcatcag 1620
cggtggagtg caatgtcatg agggaagcgg tgatcgccga agtatcgact caactatcag 1680
aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg 1740
gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga 1800
ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg 1860
cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg 1920
acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca 1980
atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc 2040
tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg 2100
atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact 2160
cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt 2220
acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc 2280
gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag 2340
aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag 2400
gcgagatcac caaggtagtc ggcaaataat tttcactatt aagaagtaat gcctactatg 2460
actcaagtcg cgaagaaaat tctagtgacg tgcgcactgc cgtacgctaa cggctcaatc 2520
cacctcggcc atatgctgga gcacatccag gctgatgtct gggtccgtta ccagcgaatg 2580
cgcggccacg aggtcaactt catctgcgcc gacgatgccc acggtacacc gatcatgctg 2640
aaagctcagc agcttggtat caccccggag cagatgattg gcgaaatgag tcaggagcat 2700
cagactgatt tcgcaggctt taacatcagc tatgacaact atcactcgac gcacagcgaa 2760
gagaaccgcc agttgtcaga acttatctac tctcgcctga aagaaaacgg ttttattaaa 2820
aaccgcacca tctctcagct gtacgatccg gaaaaaggca tgttcctgcc ggaccgtttt 2880
gtgaaaggca cctgcccgaa atgtaaatcc ccggatcaat acggcgataa ctgcgaagtc 2940
tgcggcgcga cctacagccc gactgaactg atcgagccga aatcggtggt ttctggcgct 3000
acgccggtaa tgcgtgattc tgaacacttc ttctttgatc tgccctcttt cagcgaaatg 3060
ttgcaggcat ggacccgcag cggtgcgttg caggagcagg tggcaaataa aatgcaggag 3120
tggtttgaat ctggcctgca acagtgggat atctcccgcg acgcccctta cttcggtttt 3180
gaaattccga acgcgccggg caaatatttc tacgtctggc tggacgcacc gattggctac 3240
atgggttctt tcaagaatct gtgcgacaag cgcggcgaca gcgtaagctt cgatgaatac 3300
tggaagaaag actccaccgc cgagctgtac cacttcatcg gtaaagatat tgtttacttc 3360
cacagcctgt tctggcctgc catgctggaa ggcagcaact tccgcaagcc gtccaacctg 3420
tttgttcatg gctatgtgac ggtgaacggc gcaaagatgt ccaagtctcg cggcaccttt 3480
attaaagcca gcacctggct gaatcatttt gacgcagaca gcctgcgtta ctactacact 3540
gcgaaactct cttcgcgcat tgatgatatc gatctcaacc tggaagattt cgttcagcgt 3600
gtgaatgccg atatcgttaa caaagtggtt aacctggcct cccgtaatgc gggctttatc 3660
aacaagcgtt ttgacggcgt gctggcaagc gaactggctg acccgcagtt gtacaaaacc 3720
ttcactgatg ccgctgaagt gattggtgaa gcgtgggaaa gccgtgaatt tggtaaagcc 3780
gtgcgcgaaa tcatggcgct ggctgatctg gctaaccgct atgtcgatga acaggctccg 3840
tgggtggtgg cgaaacagga aggccgcgat gccgacctgc aggcaatttg ctcaatgggc 3900
atcaacctgt tccgcgtgct gatgacttac ctgaagccgg tactgccgaa actgaccgag 3960
cgtgcagaag cattcctcaa tacggaactg acctgggatg gtatccagca accgctgctg 4020
ggccacaaag tgaatccgtt caaggcgctg tataaccgca tcgatatgag gcaggttgaa 4080
gcactggtgg aagcctctaa agaagaagta aaagccgctg ccgcgccggt aactggcccg 4140
ctggcagatg atccgattca ggaaaccatc acctttgacg acttcgctaa agttgacctg 4200
cgcgtggcgc tgattgaaaa cgcagagttt gttgaaggtt ctgacaaact gctgcgcctg 4260
acgctggatc tcggcggtga aaaacgcaat gtcttctccg gtattcgttc tgcttacccg 4320
gatccgcagg cactgattgg tcgtcacacc attatggtgg ctaacctggc accacgtaaa 4380
atgcgcttcg gtatctctga aggcatggtg atggctgccg gtcctggcgg gaaagatatt 4440
ttcctgctaa gcccggatgc cggtgctaaa ccgggtcatc aagtgaaata agatgccgct 4500
cgccagtcga ttggctgagc tcataagttc ctattccgaa gttccgcgaa cgcgtaaagg 4560
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4620
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4680
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4740
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4800
ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4860
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4920
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4980
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5040
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5100
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5160
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5220
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5280
ttcctggcct tttgctggcc ttttgctc 5308

Claims (10)

1. A protein element of a biosensor for detecting L-methionine, characterized in that: is a truncated methionyl tRNA synthetase derived from escherichia coli or a mutant thereof, wherein the amino acid sequence of the truncated methionyl tRNA synthetase derived from escherichia coli is shown as SEQ ID NO. 1; the mutant is a protein obtained by replacing at least one amino acid residue in the position L of 14, the position Y of 261 and the position H of 302 in the amino acid sequence shown in SEQ ID NO. 1.
2. The protein element of a biosensor according to claim 1, wherein: in the mutant, L at position 14 is replaced by K, G, V, D, Q, P, Y, C, F, A or W; the Y at 261 is replaced by V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F; the H at position 302 is replaced by M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N; or (b)
In the mutant, the L at position 14 is replaced by K, G, V, D, Q, P, Y, C, F, A or W, and the Y at position 261 is replaced by V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F; or (b)
The protein is a double mutant with L at position 14 replaced with K, G, V, D, Q, P, Y, C, F, A or W and H at position 302 replaced with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or any combination of N.
3. The protein element of a biosensor according to claim 2, wherein: the protein is a double mutant of any combination of substitution of Y at 261 with V, R, C, G, S, I, K, M, H, A, T or F and substitution of H at 302 with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N.
4. The protein element of a biosensor according to claim 2, wherein: the protein is a triple mutant of any combination of L at position 14 replaced with K, G, V, D, Q, P, Y, C, F, A or W, Y at position 261 replaced with V, R, C, G, S, I, K, M, H, A, T, L, P, E, Q, W, N or F, and H at position 302 replaced with M, I, T, Y, K, W, S, L, G, Q, A, C, F, V, R, P, D, E or N.
5. Nucleic acid molecule encoding a protein element of a biosensor according to any of claims 1-4, in particular said nucleic acid molecule is DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA.
6. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the nucleic acid molecule of claim 7.
7. Use of a protein element of a biosensor according to any one of claims 1 to 4, a nucleic acid molecule according to claim 7 or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising a nucleic acid molecule according to claim 8 in: 1) Preparing a biosensor for detecting L-methionine; 2) Detecting the ability of the strain to produce L-methionine; 3) Preparing a biological material for producing L-methionine or L-methionine downstream products; 4) L-methionine is produced.
8. A biosensor for detecting L-methionine, comprising an expression cassette, wherein the expression cassette comprises a wild-type promoter, an effector gene and a terminator in this order from upstream to downstream; the effector gene is a gene encoding the nucleic acid molecule of claim 5, optionally further comprising a marker protein.
9. The biosensor of claim 8, wherein: the biosensor is a plasmid containing the expression cassette of the protein element of claim 1.
10. Use of the biosensor of any of claims 8 to 9 for: detecting the ability of the strain to produce L-methionine; detecting the concentration of L-methionine in living cells of the strain; preparing a strain producing L-methionine or a downstream product of L-methionine; producing L-methionine; the starting microorganism of the biological material is escherichia coli, and more particularly the strain MG1655.
CN202210012050.6A 2022-01-07 2022-01-07 Biosensor for responding to intracellular L-methionine concentration Pending CN116445427A (en)

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