CN105755033A - General bacillus subtillis combination expression vector for improving expression level of alpha-amylase - Google Patents
General bacillus subtillis combination expression vector for improving expression level of alpha-amylase Download PDFInfo
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- CN105755033A CN105755033A CN201410784005.8A CN201410784005A CN105755033A CN 105755033 A CN105755033 A CN 105755033A CN 201410784005 A CN201410784005 A CN 201410784005A CN 105755033 A CN105755033 A CN 105755033A
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Abstract
The invention discloses a general bacillus subtillis combination expression vector for improving the expression level of alpha-amylase. The invention relates to the general bacillus subtillis combination expression vector including the following parts: a bacillus subtillis promoter sequence, a multiple cloning site sequence for combining components possibly related to expression regulation, and an escherichia coli copying initiation sequence. The vector can be stably copied in escherichia coli and can be combined with bacillus subtillis chromosomes for stable expressing. After the vector is combined with bacillus subtillis chromosomes, the expression level of alpha-amylase in bacillus subtillis is obviously improved.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of universal Bacillus subtillis integrating expression vector, be particularly well-suited to improve the Bacillus subtillis integrating expression vector of α-amylase expression.
Background technology
Bacillus subtillis (Bacillussubtilis) belong to gram positive bacteria, it is the essential industry microorganism of a kind of non-pathogenic.The work of its genome sequencing is complete.The understanding of its genetic background and physiological property is only second to escherichia coli by people.Utilize Bacillus subtillis to express foreign protein and there is plurality of advantages: (1) safety is high, can be used for the commercial production such as food, medicine；(2) can directly secretory protein be discharged in culture medium, be beneficial to separation purification；(3) having the efficient ability secreting destination protein, the recombiant protein of the eukaryotic source of its secretion in most cases still can keep native conformation and biological activity；(4) growth is rapidly, and condition of culture is simple, and genetic background is understood clear, has good industrial production base.
α-amylase (α-amylase, EC3.2.1.1), also referred to as liquefying amylase, it is with glycogen or starch for substrate, cuts α-Isosorbide-5-Nitrae glycosidic bond from intramolecule, product is dextrin, oligosaccharide containing more than four glucose residues and a small amount of glucose and maltose.For a long time, α-amylase is widely used in fields such as including starch liquefacation, yarn fabric desizing, brewage and bake always.
Wild Bacillus subtilis expression system is utilized to produce the usual yield of α-amylase very low, this is owing to α-amylase exists a lot of bottleneck in Protein transport with secretion process, on the one hand when the controlling element relating to Protein transport in cell can not meet demand, α-amylase can be caused can not to be transported to suitable cell area in Protein transport process timely and by intracellular protein enzymatic degradation, when cell relates to the restricted factor shortage of protein excretion on the other hand, affect α-amylase correctly folding in secretion process, thus causing α-amylase to be degraded by protease between cell wall.Therefore by increasing the various restricted regulatory factor relating to protein expression secretion in Bacillus subtillis, it is beneficial to the lifting of α-amylase expression-secretion amount.
Summary of the invention
It is an object of the invention to provide a kind of universal Bacillus subtillis genome conformity type expression vector, this carrier is by being incorporated into the various restricted factors that may relate to exogenous protein expression secretion bottleneck on Bacillus subtillis chromosome amyE gene by homologous recombination, it is possible to increase substantially the expression of foreign protein especially α-amylase.
The described plasmid vector of the present invention, includes:
The grac promoter sequence of Bacillus subtillis, its nucleotides sequence is classified as SEQIDNO:1.
For inserting the multiple clone site MCS of exogenous genetic fragment, its nucleotides sequence is classified as SEQIDNO:2.
For integrating gene 5 ' and 3 ' the homology arm sequences in amyE site, its nucleotide sequence respectively SEQIDNO:3 and SEQIDNO:4.
The carrier of the present invention, also includes escherichia coli replicon and chloramphenicol resistance gene coded sequence.
The carrier of the present invention, its genetic map is as in figure 2 it is shown, nucleotides sequence is classified as SEQIDNO:5.
The process LAN controlling element PrsA that the present invention relates to, its nucleotides sequence is classified as SEQIDNO:6.
The plasmid in specific embodiment 3 in the present invention, its genetic map is as it is shown on figure 3, nucleotides sequence is classified as SEQIDNO:7.
In the present invention specific embodiment 4 in the nucleotides sequence of various controlling element SecA, SecDF, SecG, Ffh, SipU, SipV, SipW be classified as SEQIDNO8-14.
The integration vector that the present invention builds is for Bacillus subtillis (such asB.subtilis168, WB600, WB700, WB800,1A751, DB104 etc.) the middle expression improving foreign protein particularly α-amylase.
The expression vector that the present invention builds, the mode specific integration of homologous recombination can be passed through to the chromosomal amyE gene location of Bacillus subtillis, by the various restricted factor that may relate to exogenous protein expression secretion bottleneck of overexpression in cell, improve foreign protein especially α-amylase transhipment level in Bacillus subtillis or promote the correct folding of albumen, thus increasing substantially foreign protein especially α-amylase secretion level in the medium.
Accompanying drawing illustrates:
Fig. 1: the structure schematic diagram of the plasmid vector pPgrac-MCS of the present invention；
Fig. 2: the genetic map of the plasmid vector pPgrac-MCS of the present invention；
Fig. 3: the genetic map of pPgrac-PrsA in embodiment 3；
Fig. 4: the bacterium colony PCR proof diagram in embodiment 3；Wherein: M.TIANGENDNAMarker III；1. bacterium colony PCR result；2. positive control；
Fig. 5: the SDS-PAGE of fermented liquid supernatant in embodiment 3: wherein: M.PageRulerTMPrestainedProteinLadder；1.1A751 fermented liquid supernatant；2.1A751(amyE::Pgrac-PrsA) fermented liquid supernatant；3.pMA5-AmyL (1A751) fermented liquid supernatant；
4.pMA5-AmyL (1A751 (amyE::Pgrac-PrsA)) fermented liquid supernatant.
Detailed description of the invention
The carrier of the present invention is with pDL for the plasmid that sets out, and in building process, is connected by double digestion and the mode of point mutation introduces Bacillus subtillis promoter grac sequence and multiple clone site, original bgaB sequence is replaced.This carrier can pass through the mode of homologous recombination and be incorporated into efficiently on Bacillus subtillis chromosome amyE position.Bacillus subtillis after integration can secrete the restricted factor of bottleneck by the various exogenous protein expression that may relate to of overexpression in cell, thus improving foreign protein secretion level in Bacillus subtillis.
Vector construction process and application to the present invention are described in detail below.
The structure of embodiment 1 plasmid vector pPgrac-MCS.
The first step, is derived from BGSC at pDL(, is numbered ECE144) multiple clone siteEcoRI andBamBacillus subtillis Pgrac promoter (comprising groE promoter, lacO handles son and gsiBCD ribosome binding site sequence) is introduced in the middle of HI.
Design primer, wherein black overstriking font component is restriction enzyme site:
Pgrac-F:5’-CCGGAATTCGAAAAGAATGATGTAAGC-3 ' (comprises EcoRI restriction enzyme site),
Pgrac-R:5’-CGCGGATCCTTCCTCCTTTAATTGG-3 ' (comprises BamHI restriction enzyme site).
With pHT43 for template, utilizing above-mentioned primer to carry out PCR, in the following order, mixed by each composition in sterilizing thin wall centrifugal tube, reaction system is:
Pgrac-F(10 μm of ol/L of forward primer) 2 μ L,
Pgrac-R(10 μm of ol/L of downstream primer) 2 μ L,
DNA profiling 1 μ L,
PrimeSTARMaxPremix (2 ×) 10 μ L,
ddH2O5 μ L,
Cumulative volume 20 μ L.
Amplification condition is: 98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 5s, 30 circulations；Last circulation is 72 DEG C of 4min.
PCR primer carries outEcoRI andBamHI double digestion and recovery, it is thus achieved that purpose fragment P1；Carrier pDL is carried outEcoRI andBamHI double digestion, reclaims purpose fragment P2.P1 carries out 16 DEG C with P2 and overnight connects and convert bacillus coli DH 5 alpha, it is thus achieved that plasmid, called after pDL-Pgrac.
Second step, on the carrier basis of above-mentioned acquisition plasmid pDL-Pgrac, introduces multiple clone site by the mode of point mutation, builds pPgrac-MCS integration vector.
Design primer, wherein black overstriking font component is restriction enzyme site:
MCS-F:5’-GCCGCACTCGAGCGAGCTC(5 ' ends carry out phosphorylation modification to CTGCACTGGATG-3 ', compriseXhoI,SacI restriction enzyme site),
MCS-R:5’-CGCAAGCTTGTCGAGGTACCTACGTAGGATCC(5 ' ends carry out phosphorylation modification to TTCCTC-3 ', compriseHindIII,KpnI,BamHI restriction enzyme site).
With plasmid pDL-Pgrac for template, carrying out PCR, in the following order, mixed by each composition in sterilizing thin wall centrifugal tube, reaction system is:
MCS-F:(10 μm of ol/L of forward primer) 2 μ L,
MCS-R:(10 μm of ol/L of downstream primer) 2 μ L,
DNA profiling 1 μ L,
PrimeSTARMaxPremix (2 ×) 10 μ L,
ddH2O5 μ L,
Cumulative volume 20 μ L.
Amplification condition is: 98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 4min, 30 circulations；Last circulation is 72 DEG C of 4min.
PCR primer utilizesDpnI carries out 37 DEG C of enzyme action 3h for digesting template plasmid, and reaction system is as follows:
PCR primer 17 μ L,
DpnI(NEB) 1 μ L,
10 × Cutsmartbuffer2 μ L,
Cumulative volume 20 μ L.
Purpose fragment after enzyme action reclaims, and T4 ligase 16 DEG C overnight connects and converts bacillus coli DH 5 alpha, it is thus achieved that plasmid, the plasmid called after pPgrac-MCS checking order correct.
Embodiment 2 builds plasmid vector pPgrac-PrsA and is integrated on Bacillus subtillis chromosome amyE.
Between cell wall, molecular chaperones PrsA(nucleotides sequence is classified as SEQIDNO:6) α-amylase can be helped quickly correctly to fold, reduce the chance that α-amylase is easily degraded by proteases between cell wall, it is possible to promote the lifting of α-amylase expression-secretion amount.The impact of the expression-secretion of α-amylase is evaluated by the carrier utilizing the present invention in PrsA process LAN situation in Bacillus subtillis.
(1) plasmid vector pPgrac-PrsA is built.
Based on pPgrac-MCS plasmid, build pPgrac-PrsA integration vector.
Design primer, wherein black overstriking font component is restriction enzyme site:
PrsA-F:5’-CGGGGTACCATGAAGAAAATCGCAATAGCAGCT-3 ' (comprisesKpnI restriction enzyme site),
PrsA-R:5’-TGAGAGCTCTTAGCATATTATGTTGCCAACTGT-3 ' (comprisesSacI restriction enzyme site),
With Bacillus subtillisBacillussubtilis168 genomes are template, carry out PCR, in the following order, are mixed by each composition in sterilizing thin wall centrifugal tube, and reaction system is:
PrsA-F(10 μm of ol/L of forward primer) 2 μ L,
PrsA-R(10 μm of ol/L of downstream primer) 2 μ L,
DNA profiling 1 μ L,
PrimeSTARMaxPremix (2 ×) 10 μ L,
ddH2O5 μ L,
Cumulative volume 20 μ L.
Amplification condition is: 98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 1min, 30 circulations；Last circulation is 72 DEG C of 4min.
PCR primer carries outKpnI andSacI double digestion and recovery, it is thus achieved that purpose fragment P3；Carrier pDL-MCS is carried outKpnI andSacI double digestion, reclaims purpose fragment P4.P3 carries out 16 DEG C with P4 and overnight connects and convert bacillus coli DH 5 alpha, it is thus achieved that plasmid, called after pPgrac-PrsA.
(2) pPgrac-PrsA is incorporated on Bacillus subtillis chromosome amyE.
Prepare as follows bacillus subtilis 1A751(laboratory preserve) competent cell:
1. the mono-bacterium colony of the 1A751 of the fresh activation of picking accesses 5mLGMI(10 × Spizizen salt ((NH4)2S042g, K2HPO418.3g, KH2PO46g, sodium citrate 1.2g, deionized water is settled to 100mL) 0.5mL, 2% acid hydrolysis casein 0.1mL, 5% yeast extract 0.1mL, 40% glucose 0.1mL, 20% magnesium sulfate monohydrate 5 μ L, 0.5%L-tryptophan 50 μ L, sterilized water polishing is to 5mL) in culture medium, 14-16h are cultivated in 37 DEG C of 200rpm concussions；
2. taking above-mentioned 500 μ L bacterium solution and be transferred in 4.5mLGMI culture medium, 4.5h is cultivated in 37 DEG C of 200rpm concussions；
3. take above-mentioned 750 μ LGMI bacterium solution to be inoculated in 4.25mLGMII(wherein 5mLGMII and comprise 10 × Spizizen salt 0.5mL, 2% acid hydrolysis casein 50 μ L, 40% glucose 0.1mL, 20% magnesium sulfate monohydrate 40 μ L, sterilized water polishing is to 5mL) in culture fluid, 1.5h is cultivated in 37 DEG C of 240rpm concussions, prepares competent cell.
Take 1 μ g plasmid pPgrac-PrsA to usePstAfter I linearization for enzyme restriction, convert Bacillus subtillis 1A751, utilize the plate screening positive colony containing chloromycetin (25 μ g/mL) resistance, be verified by colony polymerase chain reaction (PCR) method.
Bacterium colony PCR primer:
AmyE-PrsA-F:5’-GGAAGGATCCTACGTAGGT-3’
AmyE-PrsA-R:5’-ACTGTCGGAACGAGACTTC-3’
Reaction system is
AmyE-PrsA-F(10 μm of ol/L of forward primer) 2 μ L,
AmyE-PrsA-R(10 μm of ol/L of downstream primer) 2 μ L,
DNA profiling 1 μ L,
PrimeSTARMaxPremix (2 ×) 10 μ L,
ddH2O5 μ L,
Cumulative volume 20 μ L.
Amplification condition is: 98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 1min, 30 circulations；Last circulation is 72 DEG C of 4min.
PCR primer stripe size and expection consistent (accompanying drawing 4), send order-checking by bacterium colony PCR primer, and sequencing result display Pgrac-PrsA has been incorporated into amyE gene internal, is 1A751(amyE::Pgrac-PrsA by the Strain Designation built).
Embodiment 3 utilizes Bacillus subtillis 1A751(amyE::Pgrac-PrsA) fermenting and producing α-amylase.
By existing for the laboratory plasmid pMA5-AmyL(AmyL gene source carrying B. licheniformis amylase gene in bacillus licheniformis CICC10181) convert 1A751 and 1A751 (amyE::Pgrac-PrsA) respectively, one transformant of picking is inoculated in 2 × SR culture medium (3% peptone respectively, 6% yeast extract, 0.6%K2HPO4) in, 37 DEG C of overnight incubation.Within second day, it is inoculated in 30mL2 × SR culture medium culturing liquid according to 1% inoculum concentration, 37 DEG C of 200rpm fermentation culture 48h, by centrifugal for 4 DEG C of 12000rpm of fermentation liquid 10min, take supernatant detection amylase enzyme and live, each fermented sample arrange two parallel, result is as shown in table 1.
α-amylase Enzyme activity assay method carries out with reference to National Standard Method (GBT24401-2009 alpha Amylase preparation detection method).
Enzyme activity defines: when regulation, 1h liquefies 1g soluble starch, is 1 enzyme activity unit, with " U/g(U/mL) " represent.Assay method is as follows: 1. prepared by enzyme liquid: become enzymatic solution with buffer so that it is final enzyme concentration controls within 65U/mL-70U/mL scope.2. measure: (1) draws soluble starch solution (20g/L) 20mL and phosphate buffer (corresponding pH6.0) 5mL in test tube.70 DEG C of waters bath with thermostatic control preheat 8min.(2) add the enzyme liquid 1.00mL to be measured diluted, timing immediately, shake up, accurate response 5min.(3) draw the immigration of 1.00mL reactant liquor immediately and be pre-loaded with in the test tube of the rare iodine liquid of 0.5mL0.1mol/LHCl and 5mL to shake up.(4) make blank with the mixed liquor of the rare iodine liquid of 0.1mol/LHCl0.5mL and 5mL, under 660nm wavelength, measure rapidly absorbance (A) with 10mm cuvette.Table look-up according to absorbance, try to achieve the concentration (C) of tested enzyme liquid.3. calculate: in the formula of X=C × N × 16.67: the enzyme activity U/mL(U/g of X-sample)；The enzyme liquid concentration U/mL of C-test；The extension rate of N-sample；16.67-conversion constant.Acquired results represents to integer.
Table 1
From table 1 experimental result: this plasmid integration of pPgrac-PrsA to Bacillus subtillis 1A751 chromosome amyE after, the enzyme of α-amylase is lived and is significantly improved.Compared with positive control group (i.e. wild type 1A751), the enzyme work after the fermentation of test group shaking flask level can reach 750U/mL, improves 2.42 times.
Fermentation liquid after fermentation 48h is carried out SDS-PAGE electrophoresis detection, and testing result is as shown in Figure 4.
From Fig. 4 electrophoresis detection result: the expression of the α-amylase contained in fermented liquid supernatant in test group will apparently higher than positive control group, namely pPgrac-PrsA plasmid integration to Bacillus subtillis 1A751 chromosome amyE after, the secretion effect of being significantly improved to α-amylase.
Embodiment 4 is applied the carrier of the present invention and the impact of the expression-secretion of α-amylase is evaluated by the overexpression in Bacillus subtilis cells such as SecA, SecDF, SecG, Ffh, SipU, SipV, SipW.
Except molecular chaperones PrsA between the cell wall in specific embodiment 3, cell there is also the restricted factor that a lot may relate in Protein transport secretion process, specifically include following a few class, respectively (1) SRP(signal peptide identification granule) series: Ffh, HBsu, scRNA etc.；(2) signal peptidase series: SipS, SipT, SipU, SipV, SipW etc.；(3) transport protein series: SecA, SecYEG, SecDF etc.；(4) molecular chaperones series: GroES, GroEL, DnaK etc..By the various controlling element factors that may relate to are building up on the Bacillus subtillis integration vector of the present invention, it are incorporated on Bacillus subtillis 1A751 chromosome and carry out process LAN.By existing for laboratory plasmid pMA5-AmyL(AmyL gene source in bacillus licheniformis CICC10181) convert the various Bacillus subtillis bacterial strain built, and the enzyme of detection α-amylase of fermenting is lived.The expression-secretion of AmyL in this embodiment is had facilitation in various degree by part controlling element (SecA, SecDF, SecG, Ffh, SipU, SipV, SipW etc., its nucleotides sequence is classified as SEQIDNO:8-14).
(1) structure of plasmid vector.
Based on pPgrac-MCS plasmid, build pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Ffh, pPgrac-SipU, pPgrac-SipV, pPgrac-SipW integration vector.
Choose suitable multiple clone site, design primer, with Bacillus subtillisBacillussubtilis168 genomes are template, carry out PCR, in the following order, are mixed by each composition in sterilizing thin wall centrifugal tube, and reaction system is:
Forward primer (10 μm of ol/L) 2 μ L,
Downstream primer (10 μm of ol/L) 2 μ L,
DNA profiling 1 μ L,
PrimeSTARMaxPremix (2 ×) 10 μ L,
ddH2O5 μ L,
Cumulative volume 20 μ L.
Amplification condition is: 98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 15s, 72 DEG C of 2kb/min, 30 circulations；Last circulation is 72 DEG C of 4min.
PCR primer carries out double digestion and recovery, it is thus achieved that purpose fragment P5；Carrier pPgrac-MCS is carried out the double digestion of corresponding restriction enzyme site, reclaims purpose fragment P6.P5 carries out 16 DEG C with P6 and overnight connects and convert bacillus coli DH 5 alpha, it is thus achieved that plasmid, is respectively designated as pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Fth, pPgrac-SipU, pPgrac-SipV and pPgrac-SipW.
(2) the above-mentioned plasmid integration built is to Bacillus subtillis chromosome amyE.
Bacillus subtilis 1A751 competent cell is prepared according to the preparation method in specific embodiment 3.Take the 1 μ g plasmid built and choose after suitable restriction enzyme site carries out single endonuclease digestion, convert Bacillus subtillis 1A751, utilize the plate screening positive colony containing chloromycetin (25 μ g/mL) resistance, design primer, be verified by colony polymerase chain reaction (PCR) method.By verify correct bacterium colony PCR primer send order-checking, through verifying that correct bacterial strain is respectively designated as 1A751(amyE::Pgrac-SecA), 1A751(amyE::Pgrac-SecDF), 1A751(amyE::Pgrac-SecG)、1A751(amyE::Pgrac-Ffh), 1A751(amyE::Pgrac-SipU), 1A751(amyE::Pgrac-SipV) and 1A751(amyE::Pgrac-SipW).
(3) the Bacillus subtillis strain fermentation built in the present embodiment is utilized to produce α-amylase.
The existing plasmid pMA5-AmyL(AmyL gene source of laboratory is in bacillus licheniformis CICC10181) convert 1A751 and other integration bacterial strains built respectively, one transformant of picking is inoculated in 2 × SR culture medium (3% peptone respectively, 6% yeast extract, 0.6%K2HPO4) in, 37 DEG C of overnight incubation.Within second day, it is inoculated in 30mL2 × SR culture fluid according to 1% inoculum concentration, 37 DEG C of 200rpm fermentation culture 48h, by centrifugal for 4 DEG C of 12000rpm of fermentation liquid 10min, take supernatant detection amylase enzyme and live, result is as shown in table 2.
α-amylase Enzyme activity assay method reference: GBT24401-2009 alpha Amylase preparation detection method.
Concrete grammar is with reference to the method in specific embodiment 3.
Table 2
From table 2 experimental result: the plasmid integrations such as pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Fth, pPgrac-SipU, pPgrac-SipV, pPgrac-SipW to Bacillus subtillis 1A751 chromosome amyE after, the secretion of α-amylase is all had certain facilitation.Compared with positive control group (i.e. wild type 1A751), the enzyme work after the fermentation of shaking flask level can reach (287-345) U/mL, has been respectively increased 0.2-0.5 times.
Therefore the Bacillus subtillis integrating vector that the present invention builds has certain versatility, can pass through to select suitable restriction enzyme site, the various restricted factors that may relate to Protein transport and expression-secretion relevant are inserted in this carrier by the mode that enzyme action connects, thus being incorporated on Bacillus subtillis chromosome amyE, by the process LAN of various controlling elements, promote the level of expression and secretion of foreign protein particularly α-amylase.
SEQUENCELISTING
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>a kind of universal Bacillus subtillis integrating expression vector improving α-amylase expression
<130>2014
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tgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtctt840
cccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtg900
gcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccag960
ccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatat1020
ggggattggtggcgacgactcctggagcccgtcagtatcggcggaattacagctgagcgc1080
cggtcgctaccattaccagttggtctggtgtcaaaaataataataaccgggcaggccatg1140
tctgcccgtatttcgcgtaaggaaatccattatgtactatttaattctgcgtgacatccc1200
atcgatcagaccagtttttaatttgtgtgtttccatgtgtccagtttggaatactcttaa1260
cctcattggaaatcgcggcataatcactggtggtatgattgatgaccgcgtcaacaatga1320
cctttatgccatattcttcagcggctgcacacatttctttaaattcttgttcagtaccta1380
agtaacggttgccaatttgatacgatgtcggctgatacagccagtaccagttcgacatgc1440
ttttatctccttgattcccttcctttacttggttaatcggagatgtctgaatggctgtat1500
atcctgcatcatgaatatccttcatattgtgttttaacgtattgaacgaccaattccatg1560
catgaagaatggttccgcttttgatcgacggtgctgtaagctcattcgatttgttcgccg1620
tttcagcactcgcagccgccggtcctgccagaaccaaatgaaacagcaataaaaatccag1680
cgaataacggcagtaaagaggttttgaatcgttttgcaaacattcttgacactccttatt1740
tgattttttgaagacttacttcggagtcaaaaatccctcttacttcattcttccgcttcc1800
tcctttcaaaccgatgtgaagactggagaattttgttaattcttgaagacgaaagggcct1860
cgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcagg1920
tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattc1980
aaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaag2040
gaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg2100
ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtt2160
gggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttt2220
tcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggt2280
attatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaa2340
tgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag2400
agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac2460
aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac2520
tcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacac2580
cacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttac2640
tctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccact2700
tctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg2760
tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagt2820
tatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat2880
aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttta2940
gattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataa3000
tctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtaga3060
aaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac3120
aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt3180
tccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagcc3240
gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaat3300
cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaag3360
acgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc3420
cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaag3480
cgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac3540
aggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgg3600
gtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcct3660
atggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgc3720
tcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttga3780
gtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagga3840
agcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg3900
catatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacact3960
ccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgac4020
gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc4080
gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcgg4140
taaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtcc4200
agctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgtta4260
agggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatg4320
ggggtaatgataccgatgaacgagagaggatgctcacgatacgggttactgatgatgaac4380
atgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggacc4440
agagaaaatcactgttcatgggggtaatgataccgatgaaacgagagaggatgctcacga4500
tacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactgg4560
cggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgtta4620
atacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaaca4680
taatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaaga4740
ccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgct4800
cgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcc4860
tcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgc4920
gccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggttt4980
gcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgt5040
tagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacg5100
caacgcggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttcca5160
tgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccaatgatcgaagttag5220
gctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcct5280
ggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataat5340
ggggaaggccatccagcctcgcgtcgcgactaagaaaatgccgtcaaatccgctcgccat5400
gacttcactaacgatgcctttgaaaatcttcaagttcttttctactaattcaaggcgtgt5460
ctcaccaggtttttggtttgctccggcgcaaatgcagacaatatcagcatccttgcaggg5520
tatgtttctctttgatgtctttttgtttgtgaagtatttcacatttatattgtgcaacac5580
ttcacaaacttttgcaagagaaaagttttgtctgatttatgaacaaaaaagaaaccatca5640
ttgatggtttctttcggtaagtcccgtctagccttgccctcaatggggaagagaaccgct5700
taagcccgagtcattatataaaccatttagcacgtaatcaaagccaggctgattctgacc5760
gggcacttgggcgctgccattattaaaaatcacttttgcgttggttgtatccgtgtccgc5820
aggcagcgtcagcgtgtaaattccgtctgcatttttagtcattggttttccaggccaaga5880
tccggtcaattcaattactcggctcccatcatgtttatagatataagcatttacctggct5940
ccaatgattcggattttgatagccgatggttttggccgacgctggatctcttttaacaaa6000
actgtatttctcggtcctcgttacaccatcactgttcgttccttttaacatgatggtgta6060
tgttttgccaaatggatctccttttccgattgtgaattgatctccatccttaaacgccgt6120
ctctggtccattattgatttgataaacggcttttgttgtattcgcatctgcacgcaaggt6180
aatcgtcagttgatcattgaaagaatgtgttacacctgttttgtaattctcaaggaaaac6240
atgaggcgcttttgcaatatcatcaggataaagcacagctacagacctggcattgatcgt6300
gcctgtcagtttaccatcgttcacttgaaatgaacccgctccagctttattgtcatacct6360
gccatcaggcaattttgttgccgtattgatagagacagaggatgaacctgcatttgccag6420
cacaacgccatgtgagccgcgctgattcataaatatctggttgtttccattcgggttcga6480
gagttcctcaggctgtccagccatcacattgtgaaatctattgaccgcagtgatagcctg6540
atcttcaaataaagcactcccgcgatcgcctatttggcttttccccgggaacctcacacc6600
atttccgcctccctcaggtctggaaaagaaaagaggcgtactgcctgaacgagaagctat6660
caccgcccagcctaaacggatatcatcatcgctcatccatgtcgacgctctcccttatgc6720
gactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgca6780
aggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccacc6840
atacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcg6900
gtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacg6960
atgcgtccggcgtagaggatctggagctgtaatataaaaaccttcttcaactaacggggc7020
aggttagtgacattagaaaaccgactgtaaaaagtacagtcggcattatctcatattata7080
aaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcat7140
gataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattac7200
ctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattga7260
tatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcatttt7320
caggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaa7380
ggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatg7440
ttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaact7500
ctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcacta7560
agaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacac7620
taatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaata7680
tctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcct7740
aaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaa7800
tccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactt7860
tatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacatt7920
aaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttct7980
ttcttatcttgataataagggtaactattgccgatgataagctgtcaaacatgagaattc8040
gaaaagaatgatgtaagcgtgaaaaattttttatcttatcacttgaaattggaagggaga8100
ttctttattataagaattgtggaattgtgagcggataacaattcccaattaaaggaggaa8160
ggatcctacgtaggtacctcgacaagcttgcggccgcactcgagcgagct8210
<210>6
<211>879
<212>DNA
<213>Bacillussubtilis
<400>6
atgaagaaaatcgcaatagcagctatcactgctacaagcatcctcgctctcagtgcttgc60
agcagcggcgacaaagaagttatcgcaaaaacagacgcaggcgatgtcacaaaaggcgag120
ctttacacaaacatgaagaaaacagctggcgcaagcgtactgacacagctagtgcaagaa180
aaagtattggacaagaagtataaagtttcggataaagaaattgacaacaagctgaaagaa240
tacaaaacgcagcttggcgatcaatatactgccctcgaaaagcaatatggcaaagattac300
ctgaaagaacaagtaaaatatgaattgctgacacaaaaagcggctaaagataacatcaaa360
gtaacagacgccgatatcaaagagtactgggaaggcttaaaaggcaaaatccgtgcaagc420
cacatccttgttgctgataaaaagacagctgaagaagtagagaaaaagctgaaaaaaggc480
gagaagtttgaagaccttgcgaaagaatactcaacagacagctctgcttcaaaaggcggg540
gatcttggctggttcgcaaaagaaggccaaatggacgaaacattcagcaaagctgcattc600
aaattaaaaacaggtgaagtcagtgatcctgtcaaaacgcaatacggctaccatatcatt660
aaaaagacagaagaacgcggcaaatatgatgatatgaaaaaagaactgaaatctgaagtg720
cttgaacaaaaattaaatgacaacgcagctgttcaggaagctgttcaaaaagtcatgaag780
aaggctgacatcgaagtaaaagataaagatctgaaagacacatttaatacatcttcaaca840
agcaacagcacttcttcatcttcaagcaattctaaataa879
<210>7
<211>9101
<212>DNA
<213>artificial sequence
<400>7
catgaagaaaatcgcaatagcagctatcactgctacaagcatcctcgctctcagtgcttg60
cagcagcggcgacaaagaagttatcgcaaaaacagacgcaggcgatgtcacaaaaggcga120
gctttacacaaacatgaagaaaacagctggcgcaagcgtactgacacagctagtgcaaga180
aaaagtattggacaagaagtataaagtttcggataaagaaattgacaacaagctgaaaga240
atacaaaacgcagcttggcgatcaatatactgccctcgaaaagcaatatggcaaagatta300
cctgaaagaacaagtaaaatatgaattgctgacacaaaaagcggctaaagataacatcaa360
agtaacagacgccgatatcaaagagtactgggaaggcttaaaaggcaaaatccgtgcaag420
ccacatccttgttgctgataaaaagacagctgaagaagtagagaaaaagctgaaaaaagg480
cgagaagtttgaagaccttgcgaaagaatactcaacagacagctctgcttcaaaaggcgg540
ggatcttggctggttcgcaaaagaaggccaaatggacgaaacattcagcaaagctgcatt600
caaattaaaaacaggtgaagtcagtgatcctgtcaaaacgcaatacggctaccatatcat660
taaaaagacagaagaacgcggcaaatatgatgatatgaaaaaagaactgaaatctgaagt720
gcttgaacaaaaattaaatgacaacgcagctgttcaggaagctgttcaaaaagtcatgaa780
gaaggctgacatcgaagtaaaagataaagatctgaaagacacatttaatacatcttcaac840
aagcaacagcacttcttcatcttcaagcaattctaaataaagaagtctcgttccgacagt900
tggcaacataatatgctaagagctcctgcactggatggtggcgctggatggtaagccgct960
ggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcc1020
tgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaacc1080
gaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggc1140
ggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccag1200
cgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcagg1260
ctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatca1320
gttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccc1380
taacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgtt1440
gcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggca1500
gcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtca1560
aatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattgg1620
cctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgca1680
agaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtc1740
agacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcga1800
attgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacag1860
tcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatg1920
gctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagt1980
atcggcggaattacagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaa2040
ataataataaccgggcaggccatgtctgcccgtatttcgcgtaaggaaatccattatgta2100
ctatttaattctgcgtgacatcccatcgatcagaccagtttttaatttgtgtgtttccat2160
gtgtccagtttggaatactcttaacctcattggaaatcgcggcataatcactggtggtat2220
gattgatgaccgcgtcaacaatgacctttatgccatattcttcagcggctgcacacattt2280
ctttaaattcttgttcagtacctaagtaacggttgccaatttgatacgatgtcggctgat2340
acagccagtaccagttcgacatgcttttatctccttgattcccttcctttacttggttaa2400
tcggagatgtctgaatggctgtatatcctgcatcatgaatatccttcatattgtgtttta2460
acgtattgaacgaccaattccatgcatgaagaatggttccgcttttgatcgacggtgctg2520
taagctcattcgatttgttcgccgtttcagcactcgcagccgccggtcctgccagaacca2580
aatgaaacagcaataaaaatccagcgaataacggcagtaaagaggttttgaatcgttttg2640
caaacattcttgacactccttatttgattttttgaagacttacttcggagtcaaaaatcc2700
ctcttacttcattcttccgcttcctcctttcaaaccgatgtgaagactggagaattttgt2760
taattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgat2820
aataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctat2880
ttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata2940
aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccct3000
tattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaa3060
agtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaa3120
cagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttt3180
taaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcgg3240
tcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagca3300
tcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataa3360
cactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttt3420
gcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagc3480
cataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaa3540
actattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga3600
ggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgc3660
tgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccaga3720
tggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatga3780
acgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcaga3840
ccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggat3900
ctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgtt3960
ccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct4020
gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgcc4080
ggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacc4140
aaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc4200
gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtc4260
gtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctg4320
aacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata4380
cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta4440
tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc4500
ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg4560
atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt4620
cctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgt4680
ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccga4740
gcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttac4800
gcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgc4860
cgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccc4920
cgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgct4980
tacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatca5040
ccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacag5100
atgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctgg5160
cttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctcc5220
gtgtaagggggatttctgttcatgggggtaatgataccgatgaacgagagaggatgctca5280
cgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaac5340
tggcggtatggatgcggcgggaccagagaaaatcactgttcatgggggtaatgataccga5400
tgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactgg5460
aacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactc5520
agggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagc5580
atcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagac5640
tttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgc5700
agcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggc5760
aaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggcc5820
aggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatgg5880
atatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctc5940
caattcttggagtggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggt6000
ggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtatagggcggcgcc6060
tacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgat6120
cagcggtccaatgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtcc6180
ctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcgggcatcccgatgcc6240
gccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgactaagaa6300
aatgccgtcaaatccgctcgccatgacttcactaacgatgcctttgaaaatcttcaagtt6360
cttttctactaattcaaggcgtgtctcaccaggtttttggtttgctccggcgcaaatgca6420
gacaatatcagcatccttgcagggtatgtttctctttgatgtctttttgtttgtgaagta6480
tttcacatttatattgtgcaacacttcacaaacttttgcaagagaaaagttttgtctgat6540
ttatgaacaaaaaagaaaccatcattgatggtttctttcggtaagtcccgtctagccttg6600
ccctcaatggggaagagaaccgcttaagcccgagtcattatataaaccatttagcacgta6660
atcaaagccaggctgattctgaccgggcacttgggcgctgccattattaaaaatcacttt6720
tgcgttggttgtatccgtgtccgcaggcagcgtcagcgtgtaaattccgtctgcattttt6780
agtcattggttttccaggccaagatccggtcaattcaattactcggctcccatcatgttt6840
atagatataagcatttacctggctccaatgattcggattttgatagccgatggttttggc6900
cgacgctggatctcttttaacaaaactgtatttctcggtcctcgttacaccatcactgtt6960
cgttccttttaacatgatggtgtatgttttgccaaatggatctccttttccgattgtgaa7020
ttgatctccatccttaaacgccgtctctggtccattattgatttgataaacggcttttgt7080
tgtattcgcatctgcacgcaaggtaatcgtcagttgatcattgaaagaatgtgttacacc7140
tgttttgtaattctcaaggaaaacatgaggcgcttttgcaatatcatcaggataaagcac7200
agctacagacctggcattgatcgtgcctgtcagtttaccatcgttcacttgaaatgaacc7260
cgctccagctttattgtcatacctgccatcaggcaattttgttgccgtattgatagagac7320
agaggatgaacctgcatttgccagcacaacgccatgtgagccgcgctgattcataaatat7380
ctggttgtttccattcgggttcgagagttcctcaggctgtccagccatcacattgtgaaa7440
tctattgaccgcagtgatagcctgatcttcaaataaagcactcccgcgatcgcctatttg7500
gcttttccccgggaacctcacaccatttccgcctccctcaggtctggaaaagaaaagagg7560
cgtactgcctgaacgagaagctatcaccgcccagcctaaacggatatcatcatcgctcat7620
ccatgtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttg7680
aggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagt7740
cccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaag7800
tggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacct7860
gtggcgccggtgatgccggccacgatgcgtccggcgtagaggatctggagctgtaatata7920
aaaaccttcttcaactaacggggcaggttagtgacattagaaaaccgactgtaaaaagta7980
cagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaa8040
tagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaag8100
atagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggta8160
gaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaa8220
taatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaac8280
cattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattcttta8340
caggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggct8400
ctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtat8460
ttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttctt8520
gttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgttt8580
gttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttat8640
taaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttactt8700
gtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaat8760
atatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgcttt8820
agttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggattt8880
gagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatg8940
ataagctgtcaaacatgagaattcgaaaagaatgatgtaagcgtgaaaaattttttatct9000
tatcacttgaaattggaagggagattctttattataagaattgtggaattgtgagcggat9060
aacaattcccaattaaaggaggaaggatcctacgtaggtac9101
<210>8
<211>2526
<212>DNA
<213>Bacillussubtilis
<400>8
atgcttggaattttaaataaaatgtttgatccaacaaaacgtacgctgaatagatacgaa60
aaaattgctaacgatattgatgcgattcgcggagactatgaaaatctctctgacgacgca120
ttgaaacataaaacaattgaatttaaagagcgtcttgaaaaaggggcgacaacggatgat180
cttcttgttgaagctttcgctgttgttcgagaagcttcacgccgcgtaacaggcatgttt240
ccgtttaaagtccagctcatggggggcgtggcgcttcatgacggaaatatagcggaaatg300
aaaacaggggaagggaaaacattaacgtctaccctgcctgtttatttaaatgcgttaacc360
ggtaaaggcgtacacgtcgtgactgtcaacgaatacttggcaagccgtgacgctgagcaa420
atggggaaaattttcgagtttctcggtttgactgtcggtttgaatttaaactcaatgtca480
aaagacgaaaaacgggaagcttatgccgctgatattacttactccacaaacaacgagctt540
ggcttcgactatttgcgtgacaatatggttctttataaagagcagatggttcagcgcccg600
cttcattttgcggtaatagatgaagttgactctattttaattgatgaagcaagaacaccg660
cttatcatttctggacaagctgcaaaatccactaagctgtacgtacaggcaaatgctttt720
gtccgcacgttaaaagcggagaaggattacacgtacgatatcaaaacaaaagctgtacag780
cttactgaagaaggaatgacgaaggcggaaaaagcattcggcatcgataacctctttgat840
gtgaagcatgtcgcgctcaaccaccatatcaaccaggccttaaaagctcacgttgcgatg900
caaaaggacgttgactatgtagtggaagacggacaggttgttattgttgattccttcacg960
ggacgtctgatgaaaggccgccgctacagtgaggggcttcaccaagcgattgaagcaaag1020
gaagggcttgagattcaaaacgaaagcatgaccttggcgacgattacgttccaaaactac1080
ttccgaatgtacgaaaaacttgccggtatgacgggtacagctaagacagaggaagaagaa1140
ttccgcaacatctacaacatgcaggttgtcacgatccctaccaacaggcctgttgtccgt1200
gatgaccgcccggatttaatttaccgcacgatggaaggaaagtttaaggcagttgcggag1260
gatgtcgcacagcgttacatgacgggacagcctgttctagtcggtacggttgccgttgaa1320
acatctgaattgatttctaagctgcttaaaaacaaaggaattccgcatcaagtgttaaat1380
gccaaaaaccatgaacgtgaagcgcagatcattgaagaggccggccaaaaaggcgcagtt1440
acgattgcgactaacatggcggggcgcggaacggacattaagcttggcgaaggtgtaaaa1500
gagcttggcgggctcgctgtagtcggaacagaacgacatgaatcacgccggattgacaat1560
cagcttcgaggtcgttccggacgtcagggagacccggggattactcaattttatctttct1620
atggaagatgaattgatgcgcagattcggagctgagcggacaatggcgatgcttgaccgc1680
ttcggcatggacgactctactccaatccaaagcaaaatggtatctcgcgcggttgaatcg1740
tctcaaaaacgcgtcgaaggcaataacttcgattcgcgtaaacagcttctgcaatatgat1800
gatgttctccgccagcagcgtgaggtcatttataagcagcgctttgaagtcattgactct1860
gaaaacctgcgtgaaatcgttgaaaatatgatcaagtcttctctcgaacgcgcaattgca1920
gcctatacgccaagagaagagcttcctgaggagtggaagcttgacggtctagttgatctt1980
atcaacacaacttatcttgatgaaggtgcacttgagaagagcgatatcttcggcaaagaa2040
ccggatgaaatgcttgagctcattatggatcgcatcatcacaaaatataatgagaaggaa2100
gagcaattcggcaaagagcaaatgcgcgaattcgaaaaagttatcgttcttcgtgccgtt2160
gattctaaatggatggatcatattgatgcgatggatcagctccgccaagggattcacctt2220
cgtgcttacgcgcagacgaacccgcttcgtgagtatcaaatggaaggttttgcgatgttt2280
gagcatatgattgaatcaattgaggacgaagtcgcaaaatttgtgatgaaagctgagatt2340
gaaaacaatctggagcgtgaagaggttgtacaaggtcaaacaacagctcatcagccgcaa2400
gaaggcgacgataacaaaaaagcaaagaaagcaccggttcgcaaagtggttgatatcgga2460
cgaaatgccccatgccactgcggaagcgggaaaaaatataaaaattgctgcggccgtact2520
gaatag2526
<210>9
<211>2214
<212>DNA
<213>Bacillussubtilis
<400>9
atgaaaaaaggacgcttgattgcgtttttccttttcgttctattgatcggcacgggcttg60
ggctactttacgaagcctgccgctaacaatattacgttaggattggatttgcaaggcgga120
tttgaggtgctgtatgatgtacagcctgtaaaaaaaggtgacaaaatcacaaaagacgtt180
ctggtcagcacagtagaggcactgaaccgccgggccaatgttctcggtgtcagcgaaccg240
aacatccaaattgaagggaataaccggattcgcgttcagctcgctggcgtgacaaaccaa300
aacagagcgcgtgaaattttggcgactgaagcgcagctttctttcagagatgcaaatgat360
aaggaactgttaaacggtgctgatctagtcgaaaacggcgctaaacaaacttatgatagc420
acaacaaatgagccaattgtcacgattaagctgaaagacgctgataaatttggtgaagtg480
accaagaaggtcatgaaaatggcgccaaacaaccagcttgtcatttggttggattatgat540
aaaggtgattcctttaagaaagaagttcaaaaagagcatcctaaatttgtatccgctcca600
aatgtaagtcaggaactaaatacaactgatgtaaaaattgaaggtcatttcacagctcaa660
gaagcgaaagatttagccagcattttaaacgcaggcgcacttcctgtgaaactgactgaa720
aagtattcgacatcagtaggcgcgcaattcggccagcaggctctccatgatacggtgttt780
gccggtattgtcggtatcgcaattattttcttatttatgcttttctattaccgtctgccg840
ggattaatcgcggtgattacgctgtctgtttatatctacattacactccagatctttgac900
tggatgaatgccgtactcacgcttccgggaattgccgctctcattttaggtgtcgggatg960
gctgttgacgccaacattattacctatgagcggattaaagaagagctcaagctaggaaag1020
tcagtccgctctgccttccgttcaggaaacagacggtcatttgcgacgatttttgacgcg1080
aatattacaaccattattgcagcggttgtgctctttatctttgggacaagctctgttaaa1140
gggtttgcgacaatgctgatcctatcgattttgacaagctttatcactgccgttttctta1200
tcgagatttctcctcgctctccttgtggaaagcagatggcttgatcggaaaaaaggctgg1260
tttggtgtcaataagaaacatatcatggatattcaggatacggatgaaaatacagagccg1320
catacgccattccaaaaatgggatttcacgagcaaacgcaaatacttctttattttctcc1380
agtgcggtcacggttgccgggattattatcctgcttgtgttcaggctgaatcttggcatt1440
gactttgcaagcggtgcacggattgaagtgcaaagcgaccataagctgacgacagagcaa1500
gttgagaaggattttgaatctctgggtatggaccctgatactgtagttctgtcaggcgaa1560
aagagcaatatcggtgttgcccgttttgtcggggtgccagataaagaaaccattgcaaaa1620
gtaaaaacgtattttaaagacaaatacggatctgatccaaatgtcagcacagtttcaccg1680
acagtcggtaaggagctggcgagaaatgcgctgtacgcagttgctatagcttctattggc1740
atcattatttacgtttcaatccgattcgaatacaaaatggcgattgctgccatcgcctca1800
ttgctatatgacgcattctttatcgtcacgttcttcagtattacaaggcttgaggtagat1860
gttacattcatcgcggccatcttgacgataatcgggtattccattaacgatacaatcgtt1920
acatttgacagggtccgcgagcatatgaaaaagcgtaagccgaaaacctttgccgatctg1980
aaccatattgtaaacctgagcctgcagcaaacctttacacgttcaattaacactgtatta2040
accgttgtgattgttgttgtgacattgctgatctttggagcatcttctattactaacttc2100
tcaattgctttattggtcgggctgttaacaggcgtttattcttctctatacattgccgca2160
caaatttggcttgcatggaaaggaagagaactgaaaaaagattcggcgcaataa2214
<210>10
<211>231
<212>DNA
<213>Bacillussubtilis
<400>10
atgcacgcagttttgattaccttattggttatcgtcagcattgcacttattattgtcgtt60
ttgcttcaatccagtaaaagtgccggattatctggtgcgatttcaggcggagcggagcag120
ctcttcgggaaacaaaaagcaagaggtcttgatttaattttgcaccgcattacggtagtg180
ctggcagtcttgtttttcgtgttaacgattgcgcttgcttatatcctatag231
<210>11
<211>1341
<212>DNA
<213>Bacillussubtilis
<400>11
atggcatttgaaggattagccgaccgactgcagcagacgatttctaaaatccgcggaaaa60
gggaaagtcagcgaacaagatgtaaaagagatgatgcgtgaggtccgtcttgcgctgctt120
gaggctgacgttaactttaaagtagtcaaggattttgtcaaaaaagtaagtgaacgcgct180
gtaggccaagacgtcatgaaaagtctgacgcccggccagcaggtcattaaagttgttcaa240
gaggaactgactgagctgatgggcggcgaagagagcaaaatcgccgtcgcaaaaaggccg300
ccgactgttattatgatggtcggtctccaaggtgccggtaaaacgacaacaagcggtaag360
cttgcgaatctgctgcgcaaaaagcataatcgcaaaccgatgctggttgctgccgatatt420
taccgcccagccgcaattaagcagctggaaacactcggcaaacagcttgatatgcctgtt480
ttctctcttggcgatcaggtcagtcctgtagaaatagctaaacaggctattgagaaagcc540
aaggaagaacattatgactacgtcattttggatacggcagggcgcttgcatatcgaccat600
gaactgatggatgaactgaccaacgtcaaagaaatcgcgaatccggaagaaattttcctg660
gttgtcgattcaatgaccggtcaggacgctgtgaatgttgccaaaagctttaatgaacag720
ctcggtttaaccggtgttgtgttgactaagctggatggagacacacgcggcggggctgcg780
ctttctattcgcgctgtcacaaacacgccaattaagtttgcaggtttgggcgaaaagctt840
gatgcgttagagccgttccatcctgaacgcatggcatcaaggattctcggcatgggcgac900
gtgctgacattgattgaaaaagcacaggccagcgttgatgaagacaaagccaaagagctg960
gaacaaaaaatgagaacgatgagcttcacattggacgattttctggagcagctcgggcaa1020
gtcagaaacatggggccgcttgatgagcttctgcaaatgatgccgggtgcaggtaaaatg1080
aagggcctgaaaaacatccaagttgatgaaaaacagctgaatcatgtggaagcaatcatc1140
aaatcaatgactgttcttgaaaaagaacagccggatattatcaatgccagccggcggaag1200
cggattgcaaaaggaagcgggacatccgtacaggaagtcaaccgtctgcttaagcagttt1260
gatgaaatgaaaaaaatgatgaagcagatgacaaacatgtcaaaaggcaagaaaaaaggg1320
tttaagctaccttttatgtaa1341
<210>12
<211>564
<212>DNA
<213>Bacillussubtilis
<400>12
ttgaatgcaaaaacaatcacgttaaagaaaaaaagaaaaatcaaaacgatcgttgtactc60
agtatcattatgatcgcagctctcatttttacgatcagattggtgttttacaagcctttt120
cttattgaaggatcatcaatggccccaacgcttaaagactcagaaagaattctggttgat180
aaagcagtcaaatggactggcgggtttcacagaggagacatcatagtcattcatgacaaa240
aagagcggccgctcatttgtcaaacgtttaatcggtttgcctggtgacagcattaaaatg300
aaaaatgatcagctatacataaatgataaaaaggtggaagaaccatacttaaaggaatat360
aaacaggaggtcaaagagtcgggtgtaaccttaacaggtgacttcgaagttgaggttcct420
tccggtaaatattttgtgatgggagataaccgtctcaattcactggatagcagaaacgga480
atgggcatgccttctgaggacgatatcatcggtactgaatctctcgtcttttatccattc540
ggtgagatgagacaggcaaaataa564
<210>13
<211>507
<212>DNA
<213>Bacillussubtilis
<400>13
atgaaaaaacggttttggtttcttgccggtgtagtgtccgttgttctcgccattcaggtt60
aaaaatgctgtctttattgattacaaggtagaaggcgtcagtatgaacccgaccttccag120
gaaggaaacgaattgttggtcaataaattttcgcatcgatttaaaaccatccatcgtttt180
gacatcgtcctttttaaaggccctgatcataaagtgctgattaaacgggtaatcggcttg240
cccggtgaaacgatcaaatataaagatgatcagctgtatgtgaacggaaagcaggttgct300
gagccatttttgaagcatttgaaatctgtttctgccggcagccatgtaacgggtgatttt360
tctttgaaagatgtgacgggaacaagcaaggtgccgaaaggaaaatattttgtcgttgga420
gataatcgcatatacagcttcgacagccggcattttggtccgataagagaaaaaaatatt480
gtcggtgtgatttctgatgccgaataa507
<210>14
<211>573
<212>DNA
<213>Bacillussubtilis
<400>14
atgaagctgatcagtaatattttatacgtgatcatctttactcttattattgtgctgaca60
cttgtcgtgatttcaacacgttcatccgggggagagccggcagtgtttgggtatacgctg120
aaatcagttctgtcaggttcgatggagccggagttcaatacaggttccttaatattggtc180
aaagaaatcactgatgtgaaagagctccaaaaaggtgacgttattacatttatgcaggat240
gcaaatacggcggtcacccacagaattgttgacataacaaagcaaggagaccatttgtta300
tttaaaacaaaaggtgataataatgcagcagctgattcagcgcctgtatcggacgaaaat360
gttcgcgcgcaatacacaggttttcagcttccatatgccggctatatgcttcattttgcc420
agccagccgattggaacggctgtattattgattgttcccggcgtgatgctgttagtttac480
gcttttgtgacgatcagcagcgccattagagaaattgaaagaaagacaaaagccttggaa540
acagatacaaaggacagcaccatgtctacttaa573
Claims (6)
1. the Bacillus subtillis integrating expression vector improving α-amylase expression, it is characterised in that its carrier sequence is sequence 5.
2. a Bacillus subtillis integrating expression vector as claimed in claim 1, it is characterized in that: the grac promoter sequence comprising Bacillus subtillis, for integrating the multiple clone site sequence that may relate to expression regulation related elements in various Bacillus subtillis, for integrating gene 5 ' and 3 ' the homology arm sequences in amyE site, also include escherichia coli replicon and chloramphenicol resistance gene coded sequence.
3. Bacillus subtillis integrating expression vector according to claim 2, it is characterized in that the grac promoter sequence of Bacillus subtillis, for inserting the multiple clone site sequence of various expression regulation element fragment, for integrating gene 5 ' and 3 ' the homology arm sequences in amyE site, its nucleotide sequence respectively sequence 1, sequence 2, sequence 3 and sequence 4.
4. Bacillus subtillis integrating expression vector according to claim 1, it is characterised in that the host cell of its integration is Bacillus subtillis.
5. a kind of express alpha-diastatic engineering strain according to claim 4, it is specially genome amyE integrations and has the B. subtilis host cell of cell wall molecular chaperones PrsA gene (GenebankAccessionNumberX57271.1) with grac promoter, the expression-secretion of α-amylase is had obvious facilitation.
6. the Bacillus subtillis engineering strain being integrated with other restricted factors (SecA, SecDF, SecG, Ffh, SipU, SipV, SipW) relating to exogenous protein expression secretion bottleneck according to claim 4, has facilitation in various degree to the expression-secretion of α-amylase.
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| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN110564662A (en) * | 2019-09-30 | 2019-12-13 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
| CN113366113A (en) * | 2019-01-30 | 2021-09-07 | 诺维信公司 | Co-expression of homologous folding enzymes |
| CN114875057A (en) * | 2022-06-14 | 2022-08-09 | 中农华威生物制药(湖北)有限公司 | Construction method of bacillus subtilis capable of efficiently expressing feeding low-temperature acidic alpha-amylase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN113366113A (en) * | 2019-01-30 | 2021-09-07 | 诺维信公司 | Co-expression of homologous folding enzymes |
| CN110564662A (en) * | 2019-09-30 | 2019-12-13 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
| CN110564662B (en) * | 2019-09-30 | 2022-03-25 | 南京农业大学 | Construction method of integrated bacillus subtilis for efficiently expressing acetaldehyde dehydrogenase |
| CN114875057A (en) * | 2022-06-14 | 2022-08-09 | 中农华威生物制药(湖北)有限公司 | Construction method of bacillus subtilis capable of efficiently expressing feeding low-temperature acidic alpha-amylase |
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