CN111321163B - Construction and application of bacillus subtilis linear plasmid system - Google Patents
Construction and application of bacillus subtilis linear plasmid system Download PDFInfo
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Abstract
The invention discloses construction and application of a bacillus subtilis linear plasmid system, and belongs to the field of bacillus subtilis genetic engineering. The invention takes Bacillus subtilis as an expression host, and realizes the intracellular replication and expression of linear plasmids by expressing a genome replication related gene cluster derived from a Bacillus subtilis bacteriophage phi29 in the Bacillus subtilis. By replacing different promoters, the copy number and expression level of the linear plasmid can be controlled. By verification, the linear plasmid system is applied to express the green fluorescent protein, and the condition of plasmid loss does not occur after 70h of shake flask culture, and the novel expression tool lays a foundation for realizing efficient and stable gene expression, gene editing, metabolic engineering modification and the like of the bacillus subtilis.
Description
Technical Field
The invention relates to construction and application of a bacillus subtilis linear plasmid system, belonging to the field of bacillus subtilis genetic engineering.
Background
Bacillus subtilis is widely distributed in nature, including soil surfaces, aqueous environments and animal stomachs. As a model microorganism for gram-positive bacteria for laboratory studies of sporulation mechanisms and metabolic regulation. It is a non-pathogenic microorganism, free of endotoxins, and generally recognized by the U.S. Food and Drug Administration (FDA) as a safe (GRAS) food grade microorganism. In addition, Bacillus subtilis has a number of advantages, including: the growth speed is high, the culture time is short, and the culture requirement is low; the system for secreting the signal peptide is very efficient, so that the protein secretion capacity of the system is high, and great convenience is provided for a subsequent protein extraction process; the molecular chaperone system is highly efficient, so that the protein can still keep the original conformation and activity when the molecular chaperone system expresses most of proteins from eukaryotic sources. The bacillus subtilis has the advantages of wide application in the fermentation industry, and can be widely used for industrial enzyme production and biosynthesis of high value-added products. The advantages of the expression system in secreting and expressing active protein are gradually attracting attention, and the expression system is likely to become an important prokaryotic expression system following escherichia coli. However, the tool plasmid for increasing the expression level of protein is very unstable and is easy to lose, and antibiotic maintenance is required (Zhang XZ, Cui ZL, Hong Q, Li SP. high-level expression and section of methyl partial cellulose in Bacillus subtilis WB800.applied and environmental microbiology.2005; 71(7):4101-3), so the Bacillus subtilis strain with the tool plasmid is difficult to use in industrial production and is not suitable for food and pharmaceutical production.
The development of new generation tool vectors of the bacillus subtilis is not reported at present, two types of circular plasmids and expression on genomes are mainly used for gene expression at present, and the development of a novel effective linear plasmid system can lay a foundation for the fields of subsequent enzyme engineering, metabolic engineering and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a linear plasmid tool suitable for bacillus subtilis and application thereof, and the replication and expression of the linear plasmid in cells are realized by expressing a genome replication related gene cluster derived from a bacillus subtilis bacteriophage phi29 in the bacillus subtilis.
The first purpose of the invention is to provide a linear plasmid, which sequentially comprises a left replication origin, a promoter, a target gene and a right replication origin, wherein the left replication origin is shown as SEQ ID NO.3, and the right replication origin is shown as SEQ ID NO. 4.
In one embodiment, the promoter is promoter P 43 。
In one embodiment, the nucleotide sequence of the linear plasmid is set forth in SEQ ID No. 5.
The second object of the present invention is to provide an expression system of (a) or (b), wherein the target gene is expressed by a linear plasmid; wherein the content of the first and second substances,
(a) contains the linear plasmid and a plasmid carrying a gene cluster shown in SEQ ID NO. 1;
(b) cells containing the linear plasmid and a gene cluster shown in SEQ ID NO.1 integrated on the genome.
In one embodiment, (a) is a two plasmid expression system and (b) is a single plasmid expression system.
In one embodiment, both plasmids of the dual plasmid expression system are located in the same cell.
In one embodiment, the plasmid carrying the gene cluster shown in SEQ ID NO.1 is p43 NMK.
In one embodiment, in the two plasmid expression system, the plasmid carrying the gene cluster shown in SEQ ID NO.1 has the nucleotide sequence shown in SEQ ID NO. 2.
In one embodiment, the green fluorescent protein gene expressed in the linear plasmid may be replaced with other genes of interest, including but not limited to genes encoding proteins of interest, or genes involved in the regulation of microbial metabolism.
In one embodiment, the gene of interest encoding the protein of interest on the linear plasmid includes, but is not limited to, green fluorescent protein, GenBank accession No. AF 324408.1.
It is a third object of the invention to protect cells containing said linearized plasmid.
In one embodiment, the cell is a bacillus subtilis cell.
In one embodiment, the Bacillus subtilis is Bacillus subtilis 168 delta spo0A, lox72 is a knock-out of spo0A gene in Bacillus subtilis 168.
In one embodiment, the nucleotide sequence of the spo0A gene is set forth in SEQ ID NO. 7.
The fourth purpose of the invention is to provide a construction method of the linear plasmid, after the pP43NMK recombinant plasmid with the promoter is transformed into the host, the linear plasmid is transformed into the host through an electric transformation method.
The invention also claims the application of the linear plasmid in expressing natural protein, artificial protein, metabolic pathway gene and polypeptide.
Has the advantages that: the invention constructs an expression system for regulating and controlling the expression of target genes by using a bacteriophage phi29 as a regulation switch. The system is applied to express green fluorescent protein, the situation of plasmid loss does not occur after 70h of shake flask culture, and the loss rate of the control plasmid p43NMK reaches 50%. The expression tool provided by the invention has important application value for realizing efficient and stable gene expression, gene editing and metabolic engineering modification of the bacillus subtilis.
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FIG. 1 expression levels of GFP in different strains; wherein, CK: bacillus subtilis 168 wild type with P-containing 43 A P43NMK recombinant plasmid for the promoter; p43NMK-P 43 Phi29+ LP3 Bacillus subtilis 168 wild type with P 43 Promoter P43NMK recombinant plasmid and linear plasmid LP 3; p43NMK-P lytR Phi29+ LP3 Bacillus subtilis 168 wild type with P-containing lytzR Promoter P43NMK recombinant plasmid and linear plasmid LP 3;
delta spo0A-P43NMK-P43-Phi29+ LP3 Bacillus subtilis 168 Delta spo0A lox72 with P content 43 Promoter P43NMK recombinant plasmid and linear plasmid LP 3; p43NMK-P 43 -GFP: bacillus subtilis 168 delta spo0A lox72 with plasmid P43NMK-P 43 -GFP。
Detailed Description
Culturing and fermenting recombinant bacillus subtilis seeds:
medium (g/L): tryptone 10, yeast powder 5 and NaCl 10.
The method for measuring the expression level of the green fluorescent protein comprises the following steps: to each well of a 96-well plate, 200. mu.L of the diluted fermentation broth was added, and the mixture was subjected to a Cytation3 cell imaging microplate detector (Berton instruments, Inc., USA) under an excitation wavelength: 488nm, emission wavelength: 523nm, gain: 60.
example 1 construction of recombinant plasmid pP43NMK-phi29
The recombinant plasmid is P in Pp43NMK plasmid 43 The promoter is inserted into the bacillus subtilis phi29 bacteriophage to replicate the related gene cluster sequence, so as to transform into bacillus subtilis for expression. To introduce the sequence of the gene cluster related to the replication of the Bacillus subtilis phi29 phage into P 43 After the promoter, a primer rh _ p43NMK-Phi29_ F was designed: 5'-GGTACCATTATAGATGGCAAAAATGATGCAGAGAGAAATCAC-3', rh _ p43NMK-Phi29_ R: 5'-ACGCCAAGCTTTCATCATACAAACATCTCCTTTTAAACAAACGTTTATTTGATTG-3', using bacillus subtilis phi29 phage replication-related gene cluster sequence-containing escherichia coli as a template, and obtaining a bacillus subtilis phi29 phage replication-related gene cluster sequence fragment (shown as SEQ ID No. 1) through colony PCR; design of primers
fx_p43NMK-Phi29_F：5’-GGAGATGTTTGTATGATGAAAGCTTGGCGTAATCATGGTC’，fx_p43NMK-Phi29_R：5’-CATTTTTGCCATCTATAATGGTACCGCTATCACTTTATATTTTACATAATCGC-3’,
Taking the plasmid pP43NMK as a template, and obtaining a plasmid fragment through PCR reverse amplification; finally, a recombinant plasmid is constructed by a Gibson Assembly cloning Kit (New England Biolabs), and sequencing verification confirms that the construction of the recombinant pP43NMK-Phi29 plasmid is successful. Then, the successfully constructed pP43NMK-Phi29 plasmid is transformed into the bacillus subtilis 168 to obtain the bacillus subtilis P43NMK-P 43 -Phi29。
Example 2 construction of the Linear plasmid LP3
The linearized plasmid LP3 is composed of four parts, in order from left to right: left copy origin, P 43 The promoter is combined with a GFP coding gene, a chloramphenicol resistance gene and a right replication origin. Primers were designed to amplify the left replicon:
rh1_LP3_F：5’-AAAGTAAGCCCCCACCCTCACATGATACCA-3’，
rh1_LP3_R：5’-CAGCATCTTTCCTCTGCGACACAGACGAAGCGCTA-3’；
primers were designed to amplify the green fluorescent protein gene:
rh2_LP3_F：5’-CGTCTGTGTCGCAGAGGAAAGATGCTGTTCTTGTAAATGAGTTGCTAG-3’，
rh2_LP3_R：5’-GGGAAAACCCTGGCGTTAACACTTTATGCTTCCGGCTCGTATGT-3’；
primers were designed to amplify the chloramphenicol resistance gene:
rh3_LP3_F：5’-GGAAGCATAAAGTGTTAACGCCAGGGTTTTCCCAGTCACGAC-3’，
rh3_LP3_R：5’-CCAATCATAGGAGGAATTCGAGCTCGGTACCCGGGGAT-3’；
primers were designed to amplify the right replicon:
rh4_LP3_F：5’-GGGTACCGAGCTCGAATTCCTCCTATGATTGGTTGTCTTATTACCTTACTTC-3’，
rh4_LP3_R：5’-AAAGTAGGGTACAGCGACAACATACACCATTTCC-3’；
coli containing corresponding genes is taken as a template, four fragments are respectively obtained through colony PCR, then a final LP3 template is obtained through fusion PCR, after purification, primers rh1_ LP3_ F and rh4_ LP3_ R with 5' end phosphorylation are used to obtain a linear plasmid LP3 (the nucleotide sequence is shown as SEQ ID NO. 5), and LP3 is transformed into the host bacillus subtilis P43NMK-P constructed in the embodiment 1 in an electric transformation or transformation mode 43 -Phi29 to obtain Bacillus subtilis P43NMK-P 43 -Phi29+LP3。
Example 3 recombinant Bacillus subtilis P43NMK-P lytr Construction of Phi29+ LP3
The specific embodiment is the same as examples 1-2 except that P is 43 Promoter replacement by P lytr Constructing and obtaining the bacillus subtilis P43NMK-P lytr -Phi29+LP3。
Example 4 recombinant Bacillus subtilis. DELTA. spo0A-P43NMK-P 43 Construction of Phi29+ LP3
The specific implementation manner is the same as that of examples 1-2, except that Bacillus subtilis 168 is replaced by Bacillus subtilisBacillus subtilis delta spo0A-P43NMK-P is constructed by bacillus subtilis delta spo0A lox72 43 -Phi29+LP3。
The construction method of the bacillus subtilis 168 delta spo0A, lox72, comprises the following steps:
primers were designed to amplify the left homology arm:
Sqc-Spo0A-F：5’-AGATTCTGCTGCTGGCATCGGCACTAT-3’，
Sqc-Spo0A-1R：5’-CCTGTGTGAAATTGTTATCCGCTCTTGCTACATGTTTACATTCGACAAAACCGC-3’；
primers were designed to amplify spectinomycin resistance genes:
Sqc-Spo0A-2F：5’-GTCGAATGTAAACATGTAGCAAGAGCGGATAACAATTTCACACAGGAAAC-3’，
Sqc-Spo0A-2R：5’-CAGCGCAAACTAATAAATAACGCCAGGGTTTTCCCAGTC-3’；
primers were designed to amplify the right homology arm:
Sqc-Spo0A-3F：5’-GACTGGGAAAACCCTGGCGTTATTTATTAGTTTGCGCTGATAAATAGGAGGCG-3’，
Sqc-Spo0A-R：5’-AACATCACTCTGCACTCAAGATATTCAGTCGGTAA-3’；
bacillus subtilis 168 containing corresponding genes is taken as a template, three fragments are respectively obtained through colony PCR, a final knockout frame is obtained through fusion PCR, and the three fragments are transformed into a wild host Bacillus subtilis 168 in an electrotransformation or transformation mode after purification to obtain Bacillus subtilis 168 delta spo0A:: lox 72.
Example 5 measurement of expression level of GFP after transformation into Linear plasmid
Culturing Bacillus subtilis P43NMK-P in 96-well deep-well plate at 37 deg.C and 750rpm in 700 μ L LB medium 43 Transferring the seed solution of Phi29+ LP3 into 190. mu.L LB medium at an inoculum size of 5%, and culturing at 37 ℃ and 750rpm to OD 600 1. Bacillus subtilis P43NMK-P not transformed with linear plasmid under the same conditions 43 -Phi29 mean fluorescence intensity of about 333; after transformation of the linear plasmid, Bacillus subtilis P43NMK-P was detected 43 The mean fluorescence intensity of-Phi 29+ LP3 was 3591 (FIG. 1).
Example 6 plasmid loss Rate determination
The strains with the common plasmid and the linear plasmid are cultured for 10h by using LB culture medium with the temperature of 37 ℃ and the rpm of 750 mL. Then, the cells were inoculated into 50mL of LB medium at an inoculum size of 5%, and cultured at 37 ℃ and 750rpm for 70 hours. Then, after the bacterial suspension was diluted in a gradient, a chloramphenicol-free plate and a chloramphenicol-resistant plate were applied, and the number of colonies on each of the two plates was counted, and the plasmid loss rate was ═ 100% (1-number of colonies on a resistance-free plate/number of colonies on a chloramphenicol-resistant plate). The colony counts of the strain containing the linear plasmid on the two plates are almost the same, while the plasmid loss rate of the engineering strain containing the common plasmid is about 50%.
Example 7 measurement of expression level of Red fluorescent protein RFP after transformation into Linear plasmid
Construction of a linearized plasmid LP4 expressing the red fluorescent protein RFP (SEQ ID NO.6) according to the strategy of example 2, the mean fluorescence intensity of the control group not transformed with linearized plasmid was about 23 under the same culture conditions as example 3; whereas after transformation of the linear plasmid the mean value of the fluorescence intensity was 359.
Comparative example 1 construction of pP43NMK-GFP recombinant plasmid and verification
The pP43NMK vector is currently one of the most commonly used high copy number vectors in B.subtilis, and is used to express GFP as a control. After the pP43NMK-GFP recombinant plasmid is transformed into a host, seeds are cultured for 10h by using a 96-hole deep-hole plate and at 37 ℃ and 750rpm in 700 mu L of LB conditioned medium. Then, the cells were inoculated in 190. mu.L of LB medium at an inoculum size of 5%, and cultured at 37 ℃ and 750rpm to OD 600 When the mean fluorescence intensity was 5059, it was found that the protein expression level of the linear plasmid reached 70% of the expression level of the commonly used high copy carrier protein.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> construction and application of bacillus subtilis linear plasmid system
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 3777
<212> DNA
<213> Artificial sequence
<400> 1
atggcaaaaa tgatgcagag agaaatcaca aagacaaccg tcaacgttgc caaaatggtg 60
atggtggacg gagaggttca ggtagagcaa ctaccatctg aaacatttgt gggtaatctg 120
acaatggaac aggctcaatg gagaatgaag cgcaaatata aaggcgaacc tgttcaagtg 180
gtaagcgttg aacctaacac agaggtttat gagctacctg tagaaaaatt ccttgaagtt 240
gctaccgttc gggtagagaa agacgaagat caagaggaac aaacagaagc tccagaagaa 300
caggttgctg aatgatggaa acacaagaga aggttctagt attagttgga gcgctattca 360
taaacacctt caatcttaat tataaaagca ttgacctaac acacgattca gaacaagctt 420
acgggtttaa agataaatgg gaagcgcaga aagttgccgc aaatgtaggc ggacaagttg 480
tcatcagaac aaccagtttc aagatcgttt aactcttaac actctcttaa cacgcacgac 540
ctatcaaata aaccaaaatg aaaaggatga ttaataatgg aaaacacaaa catcgtaaag 600
gctacttttg acacagaaac tcttgaagga caaatcaaaa tctttaatgc tcagacaggc 660
ggcggacaat cttttaaaaa ccttccagat ggaacaatta tagaagccaa cgccattgct 720
caatataagc aagtgtccga tacatacggg gacgctaagg aagaaacagt tactactatt 780
tttgcggctg acgggtcgtt atattccgct atctctaaga ctgtagcaga agccgcatct 840
gacttaattg accttgtgac tcgtcataag cttgaaacgt ttaaggttaa agtggttcaa 900
ggaacatcta gtaaaggtaa cgtattcttt agcttacaac tatccctata aacaggaggt 960
aaaatataga tgcctaaaac acaaagaggt atctatcata acttgaagga atctgaatac 1020
gtggcatcta acaccgatgt cacgtttttc ttttcaagtg aattgtattt gaacaagttt 1080
ctcgatggat accaagaata caggaagaaa tttaataaga agatagaacg ggtcgctgtt 1140
acaccgtgga atatggatat gctcgcagac atcacgttct attcagaagt tgaaaagcgt 1200
ggtttccatg cttggttgaa aggagataac gcaacatggc gagaagtcca cgtatacgca 1260
ttaaggataa tgacaaagcc gaatacgctc gattggtcaa gaatacaaaa gccaagattg 1320
cgagaacgaa gaaaaagtat ggtgtagacc ttaccgctga aattgatata cctgaccttg 1380
attcatttga aacacgggcg cagttcaata agtggaagga acaagcgtcc tctttcacta 1440
accgtgctaa tatgcgttat cagttcgaaa agaatgcata cggtgtggtg gctagtaaag 1500
ctaagatagc tgagattgaa cgtaacacaa aagaggttca gcggttagta gatgagaaaa 1560
tcaaggctat gaaagacaaa gaatactatg caggcggtaa gccgcaaggg acaattgaac 1620
aacggatagc tatgacaagt cctgcacacg ttacaggaat taatagaccc catgattttg 1680
actttagcaa ggtgcgaagc tatagccgtt tgcgaaccct agaagaaagc atggagatga 1740
gaacagaccc tcagtattat gaaaagaaaa tgatacagtt acagttaaac tttattaaga 1800
gcgttgaggg tagtttcaat tcatttgatg cggcagatga actgatcgaa gaattaaaaa 1860
agatacctcc tgatgacttc tatgaattgt ttctcagaat atcagaaata tcctttgagg 1920
aatttgatag tgagggaaac acagtggaga acgtagaagg taatgtatat aaaatactgt 1980
catacttgga acagtatcga aggggtgact ttgatctaag cttaaagggg ttctaggctc 2040
cgttaaagga tgaagcatat gccgagaaag atgtatagtt gtgactttga gacaactact 2100
aaagtggaag actgtagggt atgggcgtat ggttatatga atatagaaga tcacagtgag 2160
tacaaaatag gtaatagcct ggatgagttt atggcgtggg tgttgaaggt acaagctgat 2220
ctatatttcc ataacctcaa atttgacgga gcttttatca ttaactggtt ggaacgtaat 2280
ggttttaagt ggtcggctga cggattgcca aacacatata atacgatcat atctcgcatg 2340
ggacaatggt acatgattga tatatgttta ggctacaaag ggaaacgtaa gatacataca 2400
gtgatatatg acagcttaaa gaaactaccg tttcctgtta agaagatagc taaagacttt 2460
aaactaactg ttcttaaagg tgatattgat taccacaaag aaagaccagt cggctataag 2520
ataacacccg aagaatacgc ctatattaaa aacgatattc agattattgc ggaagctctg 2580
ttaattcagt ttaagcaagg tttagaccgg atgacagcag gcagtgacag tctaaaaggt 2640
ttcaaggata ttataaccac taagaaattc aaaaaggtgt ttcctacatt gagtcttgga 2700
ctcgataagg aagtgagata cgcctataga ggtggtttta catggttaaa tgataggttc 2760
aaagaaaaag aaatcggaga aggcatggtc ttcgatgtta atagtctata tcctgcacag 2820
atgtatagcc gtctccttcc atatggtgaa cctatagtat tcgagggtaa atacgtttgg 2880
gacgaagatt acccactaca catacagcat atcagatgtg agttcgaatt gaaagagggc 2940
tatataccca ctatacagat aaaaagaagt aggttttata aaggtaatga gtacctaaaa 3000
agtagcggcg gggagatagc cgacctctgg ttgtcaaatg tagacctaga attaatgaaa 3060
gaacactacg atttatataa cgttgaatat atcagcggct taaaatttaa agcaactaca 3120
ggtttgttta aagattttat agataaatgg acgtacatca agacgacatc agaaggagcg 3180
atcaagcaac tagcaaaact gatgttaaac agtctatacg gtaaattcgc tagtaaccct 3240
gatgttacag ggaaagtccc ttatttaaaa gagaatgggg cgctaggttt cagacttgga 3300
gaagaggaaa caaaagaccc tgtttataca cctatgggcg ttttcatcac tgcatgggct 3360
agatacacga caattacagc ggcacaggct tgttatgatc ggataatata ctgtgatact 3420
gacagcatac atttaacggg tacagagata cctgatgtaa taaaagatat agttgaccct 3480
aagaaattgg gatactgggc acatgaaagt acattcaaaa gagctaaata tctgagacag 3540
aagacctata tacaagacat ctatatgaaa gaagtagatg gtaagttagt agaaggtagt 3600
ccagatgatt acactgatat aaaatttagt gttaaatgtg cgggaatgac tgacaagatt 3660
aagaaagagg ttacgtttga gaatttcaaa gtcggattca gtcggaaaat gaagcctaag 3720
cctgtgcaag tgccgggcgg ggtggttctg gttgatgaca cattcacaat caaataa 3777
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<211> 10490
<212> DNA
<213> Artificial sequence
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tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccttaaggaa cgtacagacg 420
gcttaaaagc ctttaaaaac gtttttaagg ggtttgtaga caaggtaaag gataaaacag 480
cacaattcca agaaaaacac gatttagaac ctaaaaagaa cgaatttgaa ctaactcata 540
accgagaggt aaaaaaagaa cgaagtcgag atcagggaat gagtttataa aataaaaaaa 600
gcacctgaaa aggtgtcttt ttttgatggt tttgaacttg ttctttctta tcttgataca 660
tatagaaata acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt 720
atgtgtttta aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta 780
aagttataag tgactaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt 840
cctaatagta gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt 900
ggaaaagtga gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg 960
catattcttg aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat 1020
aaacaaaatc gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg 1080
ctttgtccaa tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt 1140
gttgctgaag ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt 1200
aaaaatgttt atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt 1260
cgccgaatga tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg 1320
gaagtgacaa taaataataa agataattct tataatcagc acatgcatgt attggtatgt 1380
gtggaaccaa cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa 1440
ttttggaaaa aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga 1500
ccgaaaaata aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct 1560
gtaaaggata cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat 1620
ttggaggaag gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata 1680
cataaaaaat taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac 1740
gaaaaagccg atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat 1800
tattttatta aagagtagtt caacaaacgg gccagtttgt tgaagattag atgctataat 1860
tgttattaaa aggattgaag gatgcttagg aagacgagtt attaatagct gaataagaac 1920
ggtgctctcc aaatattctt atttagaaaa gcaaatctaa aattatctga aaagggaatg 1980
agaatagtga atggaccaat aataatgact agagaagaaa gaatgaagat tgttcatgaa 2040
attaaggaac gaatattgga taaatatggg gatgatgtta aggctattgg tgtttatggc 2100
tctcttggtc gtcagactga tgggccctat tcggatattg agatgatgtg tgtcatgtca 2160
acagaggaag cagagttcag ccatgaatgg acaaccggtg agtggaaggt ggaagtgaat 2220
tttgatagcg aagagattct actagattat gcatctcagg tggaatcaga ttggccgctt 2280
acacatggtc aatttttctc tattttgccg atttatgatt caggtggata cttagagaaa 2340
gtgtatcaaa ctgctaaatc ggtagaagcc caaacgttcc acgatgcgat ttgtgccctt 2400
atcgtagaag agctgtttga atatgcaggc aaatggcgta atattcgtgt gcaaggaccg 2460
acaacatttc taccatcctt gactgtacag gtagcaatgg caggtgccat gttgattggt 2520
ctgcatcatc gcatctgtta tacgacgagc gcttcggtct taactgaagc agttaagcaa 2580
tcagatcttc cttcaggtta tgaccatctg tgccagttcg taatgtctgg tcaactttcc 2640
gactctgaga aacttctgga atcgctagag aatttctgga atgggattca ggagtggaca 2700
gaacgacacg gatatatagt ggatgtgtca aaacgcatac cattttgaac gatgacctct 2760
aataattgtt aatcatgttg gttacgtatt tattaacttc tcctagtatt agtaattatc 2820
atggctgtca tggcgcatta acggaataaa gggtgtgctt aaatcgggcc attttgcgta 2880
ataagaaaaa ggattaatta tgagcgaatt gaattaataa taaggtaata gatttacatt 2940
agaaaatgaa aggggatttt atgcgtgaga atgttacagt ctatcccggc attgccagtc 3000
ggggatatta aaaagagtat aggtttttat tgggataaag taggtttcac tttggttcac 3060
catgaagatg gattcgcagt tctaatgtgt aatgaggttc ggattcatct atgggaggca 3120
agtgatgaag gctggcgcct cgtagtaatg attcaccggt ttgtacaggt gcggagtcgt 3180
ttattgctgg tactgctagt tgccgcattg aagtagaggg aattgatgaa ttatatcaac 3240
atattaagcc tttgggcatt ttgcacccca atacatcatt aaaagatcag tggtgggatg 3300
aacgagactt tgcagtaatt gatcccgaca acaatttgat tagctttttt caacaaataa 3360
aaagctaaaa tctattatta atctgttcag caatcgggcg cgattgctga ataaaagata 3420
cgagagacct ctcttgtatc ttttttattt tgagtggttt tgtccgttac actagaaaac 3480
cgaaagacaa taaaaatttt attcttgctg agtctggctt tcggtaagct agacaaaacg 3540
gacaaaataa aaattggcaa gggtttaaag gtggagattt tttgagtgat cttctcaaaa 3600
aatactacct gtcccttgct gatttttaaa cgagcacgag agcaaaaccc ccctttgctg 3660
aggtggcaga gggcaggttt ttttgtttct tttttctcgt aaaaaaaaga aaggtcttaa 3720
aggttttatg gttttggtcg gcactgccgc gcctcgcaga gcacacactt tatgaatata 3780
aagtatagtg tgttatactt tacttggaag tggttgccgg aaagagcgaa aatgcctcac 3840
atttgtgcca cctaaaaagg agcgatttac atatgagtta tgcagtttgt agaatgcaaa 3900
aagtgaaatc agctggacta aaaggcatgc aatttcataa tcaaagagag cgaaaaagta 3960
gaacgaatga tgatattgac catgagcgaa cacgtgaaaa ttatgatttg aaaaatgata 4020
aaaatattga ttacaacgaa cgtgtcaaag aaattattga atcacaaaaa acaggtacaa 4080
gaaaaacgag gaaagatgct gttcttgtaa atgagttgct agtaacatct gaccgagatt 4140
tttttgagca actggatcct gataggtggt atgttttcgc ttgaactttt aaatacagcc 4200
attgaacata cggttgattt aataactgac aaacatcacc ctcttgctaa agcggccaag 4260
gacgccgccg ccggggctgt ttgcgttctt gccgtgattt cgtgtaccat tggtttactt 4320
atttttttgc caaggctgta atggctgaaa attcttacat ttattttaca tttttagaaa 4380
tgggcgtgaa aaaaagcgcg cgattatgta aaatataaag tgatagcggt accattatag 4440
atggcaaaaa tgatgcagag agaaatcaca aagacaaccg tcaacgttgc caaaatggtg 4500
atggtggacg gagaggttca ggtagagcaa ctaccatctg aaacatttgt gggtaatctg 4560
acaatggaac aggctcaatg gagaatgaag cgcaaatata aaggcgaacc tgttcaagtg 4620
gtaagcgttg aacctaacac agaggtttat gagctacctg tagaaaaatt ccttgaagtt 4680
gctaccgttc gggtagagaa agacgaagat caagaggaac aaacagaagc tccagaagaa 4740
caggttgctg aatgatggaa acacaagaga aggttctagt attagttgga gcgctattca 4800
taaacacctt caatcttaat tataaaagca ttgacctaac acacgattca gaacaagctt 4860
acgggtttaa agataaatgg gaagcgcaga aagttgccgc aaatgtaggc ggacaagttg 4920
tcatcagaac aaccagtttc aagatcgttt aactcttaac actctcttaa cacgcacgac 4980
ctatcaaata aaccaaaatg aaaaggatga ttaataatgg aaaacacaaa catcgtaaag 5040
gctacttttg acacagaaac tcttgaagga caaatcaaaa tctttaatgc tcagacaggc 5100
ggcggacaat cttttaaaaa ccttccagat ggaacaatta tagaagccaa cgccattgct 5160
caatataagc aagtgtccga tacatacggg gacgctaagg aagaaacagt tactactatt 5220
tttgcggctg acgggtcgtt atattccgct atctctaaga ctgtagcaga agccgcatct 5280
gacttaattg accttgtgac tcgtcataag cttgaaacgt ttaaggttaa agtggttcaa 5340
ggaacatcta gtaaaggtaa cgtattcttt agcttacaac tatccctata aacaggaggt 5400
aaaatataga tgcctaaaac acaaagaggt atctatcata acttgaagga atctgaatac 5460
gtggcatcta acaccgatgt cacgtttttc ttttcaagtg aattgtattt gaacaagttt 5520
ctcgatggat accaagaata caggaagaaa tttaataaga agatagaacg ggtcgctgtt 5580
acaccgtgga atatggatat gctcgcagac atcacgttct attcagaagt tgaaaagcgt 5640
ggtttccatg cttggttgaa aggagataac gcaacatggc gagaagtcca cgtatacgca 5700
ttaaggataa tgacaaagcc gaatacgctc gattggtcaa gaatacaaaa gccaagattg 5760
cgagaacgaa gaaaaagtat ggtgtagacc ttaccgctga aattgatata cctgaccttg 5820
attcatttga aacacgggcg cagttcaata agtggaagga acaagcgtcc tctttcacta 5880
accgtgctaa tatgcgttat cagttcgaaa agaatgcata cggtgtggtg gctagtaaag 5940
ctaagatagc tgagattgaa cgtaacacaa aagaggttca gcggttagta gatgagaaaa 6000
tcaaggctat gaaagacaaa gaatactatg caggcggtaa gccgcaaggg acaattgaac 6060
aacggatagc tatgacaagt cctgcacacg ttacaggaat taatagaccc catgattttg 6120
actttagcaa ggtgcgaagc tatagccgtt tgcgaaccct agaagaaagc atggagatga 6180
gaacagaccc tcagtattat gaaaagaaaa tgatacagtt acagttaaac tttattaaga 6240
gcgttgaggg tagtttcaat tcatttgatg cggcagatga actgatcgaa gaattaaaaa 6300
agatacctcc tgatgacttc tatgaattgt ttctcagaat atcagaaata tcctttgagg 6360
aatttgatag tgagggaaac acagtggaga acgtagaagg taatgtatat aaaatactgt 6420
catacttgga acagtatcga aggggtgact ttgatctaag cttaaagggg ttctaggctc 6480
cgttaaagga tgaagcatat gccgagaaag atgtatagtt gtgactttga gacaactact 6540
aaagtggaag actgtagggt atgggcgtat ggttatatga atatagaaga tcacagtgag 6600
tacaaaatag gtaatagcct ggatgagttt atggcgtggg tgttgaaggt acaagctgat 6660
ctatatttcc ataacctcaa atttgacgga gcttttatca ttaactggtt ggaacgtaat 6720
ggttttaagt ggtcggctga cggattgcca aacacatata atacgatcat atctcgcatg 6780
ggacaatggt acatgattga tatatgttta ggctacaaag ggaaacgtaa gatacataca 6840
gtgatatatg acagcttaaa gaaactaccg tttcctgtta agaagatagc taaagacttt 6900
aaactaactg ttcttaaagg tgatattgat taccacaaag aaagaccagt cggctataag 6960
ataacacccg aagaatacgc ctatattaaa aacgatattc agattattgc ggaagctctg 7020
ttaattcagt ttaagcaagg tttagaccgg atgacagcag gcagtgacag tctaaaaggt 7080
ttcaaggata ttataaccac taagaaattc aaaaaggtgt ttcctacatt gagtcttgga 7140
ctcgataagg aagtgagata cgcctataga ggtggtttta catggttaaa tgataggttc 7200
aaagaaaaag aaatcggaga aggcatggtc ttcgatgtta atagtctata tcctgcacag 7260
atgtatagcc gtctccttcc atatggtgaa cctatagtat tcgagggtaa atacgtttgg 7320
gacgaagatt acccactaca catacagcat atcagatgtg agttcgaatt gaaagagggc 7380
tatataccca ctatacagat aaaaagaagt aggttttata aaggtaatga gtacctaaaa 7440
agtagcggcg gggagatagc cgacctctgg ttgtcaaatg tagacctaga attaatgaaa 7500
gaacactacg atttatataa cgttgaatat atcagcggct taaaatttaa agcaactaca 7560
ggtttgttta aagattttat agataaatgg acgtacatca agacgacatc agaaggagcg 7620
atcaagcaac tagcaaaact gatgttaaac agtctatacg gtaaattcgc tagtaaccct 7680
gatgttacag ggaaagtccc ttatttaaaa gagaatgggg cgctaggttt cagacttgga 7740
gaagaggaaa caaaagaccc tgtttataca cctatgggcg ttttcatcac tgcatgggct 7800
agatacacga caattacagc ggcacaggct tgttatgatc ggataatata ctgtgatact 7860
gacagcatac atttaacggg tacagagata cctgatgtaa taaaagatat agttgaccct 7920
aagaaattgg gatactgggc acatgaaagt acattcaaaa gagctaaata tctgagacag 7980
aagacctata tacaagacat ctatatgaaa gaagtagatg gtaagttagt agaaggtagt 8040
ccagatgatt acactgatat aaaatttagt gttaaatgtg cgggaatgac tgacaagatt 8100
aagaaagagg ttacgtttga gaatttcaaa gtcggattca gtcggaaaat gaagcctaag 8160
cctgtgcaag tgccgggcgg ggtggttctg gttgatgaca cattcacaat caaataaacg 8220
tttgtttaaa aggagatgtt tgtatgatga aagcttggcg taatcatggt catagctgtt 8280
tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 8340
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 8400
gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 8460
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 8520
ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 8580
cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 8640
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 8700
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 8760
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 8820
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 8880
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 8940
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 9000
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 9060
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 9120
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 9180
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 9240
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 9300
gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 9360
gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 9420
gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 9480
ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 9540
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 9600
agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 9660
ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 9720
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 9780
ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 9840
caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 9900
gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 9960
atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 10020
accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 10080
aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 10140
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 10200
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 10260
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 10320
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 10380
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 10440
tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 10490
<210> 3
<211> 191
<212> DNA
<213> Artificial sequence
<400> 3
aaagtaagcc cccaccctca catgatacca ttctcctaat atcgacataa tccgtcgatc 60
ctcggcatac catgatcagg gagggaaact actacttaat atatcaatct atagacctac 120
tagataggtt tgtcaatgaa caacataaaa cgacacagaa tcccacgttt tagcgcttcg 180
tctgtgtcgc a 191
<210> 4
<211> 194
<212> DNA
<213> Artificial sequence
<400> 4
cctcctatga ttggttgtct tattacctta cttctattat agtataacat gttaaacgat 60
agtttgtcta cccttttcga caaattgatg ataataaata gtataggtat atagtcgtga 120
tttagttgtt agattcttgt cgaagatagt cggtcaatgg ggaaatggtg tatgttgtcg 180
ctgtacccta cttt 194
<210> 5
<211> 2878
<212> DNA
<213> Artificial sequence
<400> 5
aaagtaagcc cccaccctca catgatacca ttctcctaat atcgacataa tccgtcgatc 60
ctcggcatac catgatcagg gagggaaact actacttaat atatcaatct atagacctac 120
tagataggtt tgtcaatgaa caacataaaa cgacacagaa tcccacgttt tagcgcttcg 180
tctgtgtcgc agaggaaaga tgctgttctt gtaaatgagt tgctagtaac atctgaccga 240
gatttttttg agcaactgga tcctgatagg tggtatgttt tcgcttgaac ttttaaatac 300
agccattgaa catacggttg atttaataac tgacaaacat caccctcttg ctaaagcggc 360
caaggacgcc gccgccgggg ctgtttgcgt tcttgccgtg atttcgtgta ccattggttt 420
acttattttt ttgccaaggc tgtaatggct gaaaattctt acatttattt tacattttta 480
gaaatgggcg tgaaaaaaag cgcgcgatta tgtaaaatat aaagtgatag cggtaccatt 540
ataggtaaga gaggaatgta cacatgaaaa tcaaaaacaa ccaacaaaaa aatgaactga 600
ttcaaatgag taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag 660
atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat 720
acggaaaact tacccttaaa tttatttgca ctactggaaa gcttcctgtt ccttggccaa 780
cacttgtcac tactcttact tatggtgttc aatgcttttc aagataccca gatcatatga 840
agcggcacga cttcttcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct 900
tcttcaagga cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc 960
tcgtcaacag aatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc 1020
acaagttgga atacaactac aactcccaca acgtatacat catggcagac aaacaaaaga 1080
atggaatcaa agttaacttc aaaattagac acaacattga agatggaagc gttcaactag 1140
cagaccatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc 1200
attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg 1260
tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat 1320
aatgatgaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 1380
ctcacaattc cacacaacat acgagccgga agcataaagt gttaacgcca gggttttccc 1440
agtcacgacg ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag gtcgacgatt 1500
ctaccgttcg tataatgtat gctatacgaa gttatttatt ggtatgactg gttttaagcg 1560
caaaaaaagt tgctttttcg tacctattaa tgtatcgtta gaaaaccgac tgtaaaaagt 1620
acagtcggca ttatctcata ttataaaagc cagtcattag gcctatctga caattcctga 1680
atagagttca taaacaatcc tgcatgataa ccatcacaaa cagaatgatg tacctgtaaa 1740
gatagcggta aatatattga attaccttta ttaatgaatt ttcctgctgt aataatgggt 1800
agaaggtaat tactattatt attgatattt aagttaaacc cagtaaatga agtccatgga 1860
ataatagaaa gagaaaaagc attttcaggt ataggtgttt tgggaaacaa tttccccgaa 1920
ccattatatt tctctacatc agaaaggtat aaatcataaa actctttgaa gtcattcttt 1980
acaggagtcc aaataccaga gaatgtttta gatacaccat caaaaattgt ataaagtggc 2040
tctaacttat cccaataacc taactctccg tcgctattgt aaccagttct aaaagctgta 2100
tttgagttta tcacccttgt cactaagaaa ataaatgcag ggtaaaattt atatccttct 2160
tgttttatgt ttcggtataa aacactaata tcaatttctg tggttatact aaaagtcgtt 2220
tgttggttca aataatgatt aaatatctct tttctcttcc aattgtctaa atcaatttta 2280
ttaaagttca tttgatatgc ctcctaaatt tttatctaaa gtgaatttag gaggcttact 2340
tgtctgcttt cttcattaga atcaatcctt ttttaaaagt caatattact gtaacataaa 2400
tatatatttt aaaaatatcc cactttatcc aattttcgtt tgttgaacta atgggtgctt 2460
tagttgaaga ataaaagacc acattaaaaa atgtggtctt ttgtgttttt ttaaaggatt 2520
tgagcgtagc gaaaaatcct tttctttctt atcttgataa taagggtaac tattgccgtc 2580
gtccattccg acagcatcgc cagtcactat ggcataactt cgtataatgt atgctatacg 2640
aacggtacaa tctctagagg atccccgggt accgagctcg aattcctcct atgattggtt 2700
gtcttattac cttacttcta ttatagtata acatgttaaa cgatagtttg tctacccttt 2760
tcgacaaatt gatgataata aatagtatag gtatatagtc gtgatttagt tgttagattc 2820
ttgtcgaaga tagtcggtca atggggaaat ggtgtatgtt gtcgctgtac cctacttt 2878
<210> 6
<211> 699
<212> DNA
<213> Artificial sequence
<400> 6
atggtttctg aactgatcaa agaaaacatg cacatgaaac tgtacatgga aggtaccgtt 60
aacaaccacc acttcaaatg cacctctgaa ggtgaaggta aaccgtacga aggtacccag 120
accatgcgta tcaaagctgt tgaaggtggt ccgctgccgt tcgcttttga catcctggct 180
acctctttca tgtacggttc taaaaccttc atcaaccaca cccagggtat cccggacttt 240
ttcaaacagt ctttcccgga aggtttcacc tgggaacgtg ttaccaccta cgaagacggt 300
ggtgttctga ccgctaccca ggacacctct ctgcaagacg gttgcctgat ctacaacgtt 360
aaaatccgtg gtgttaactt cccgtctaac ggtccggtta tgcagaaaaa aaccctgggt 420
tgggaagctt ctaccgaaac cctgtacccg gctgacggtg gtctggaagg tcgtgctgac 480
atggctctga aactggttgg tggtggtcac ctgatctgca acctgaaaac cacctaccgt 540
tctaaaaaac cggctaaaaa cctgaaaatg ccgggtgttt actacgttga ccgtcgtctg 600
gaacgtatca aagaagctga caaagaaacc tacgttgaac agcacgaagt tgctgttgct 660
cgttactgcg acctgccgtc taaactgggt caccgttaa 699
<210> 7
<211> 804
<212> DNA
<213> Bacillus subtilis
<400> 7
gtggagaaaa ttaaagtttg tgttgctgat gataatcgag agctggtaag cctgttaagt 60
gaatatatag aaggacagga agacatggaa gtgatcggcg ttgcttataa cggacaggaa 120
tgcctgtcgc tgtttaaaga aaaagatccc gatgtgctcg tattagatat tattatgccg 180
catctagacg gacttgcggt tttagagagg ctgagggaat cagatctgaa aaaacagccg 240
aatgtcatta tgctgacagc ctttgggcag gaagatgtca cgaaaaaggc cgtcgattta 300
ggcgcgtcct actttattct caaaccgttt gatatggaaa accttgtcgg ccatatccgc 360
caggtcagcg gaaatgccag cagtgtgacg catcgtgcgc catcatcgca aagcagtatt 420
atacgcagca gccagcctga accaaagaag aaaaatctcg acgcgagcat cacaagcatt 480
atccatgaaa tcggcgtccc agcccatatt aaaggctatc tctatctgcg cgaagcaatc 540
tcaatggtat acaatgacat cgaattgctc ggcagcatta caaaagtcct ctatccggac 600
atcgccaaaa aatttaacac aaccgcaagc cgtgtagaaa gagcgatccg ccatgcaatt 660
gaagtggcat ggagcagagg aaacattgat tccatttcct cgttgtttgg ttatactgtc 720
agcatgacaa aagctaaacc taccaacagt gaattcattg caatggttgc ggataagctg 780
aggttagagc ataaggcttc ttaa 804
Claims (9)
1. A gene expression system is characterized in that the expression system comprises a linear plasmid and (a) or (b), wherein the linear plasmid sequentially comprises a left replication origin, a promoter, a target gene and a right replication origin from a 5 'end to a 3' end, the left replication origin is shown as SEQ ID No.3, and the right replication origin is shown as SEQ ID No. 4; wherein the content of the first and second substances,
(a) is a plasmid carrying a gene cluster shown in SEQ ID NO. 1;
(b) is a cell integrating a gene cluster shown as SEQ ID NO.1 on a genome;
the cell is a Bacillus subtilis cell.
2. The gene expression system of claim 1, wherein the promoter is promoter P lytr Or P 43 。
3. The gene expression system of claim 1, wherein the nucleotide sequence of the plasmid carrying the gene cluster shown in SEQ ID No.1 is shown in SEQ ID No. 2; the sequence of the linear plasmid is shown as SEQ ID NO. 5.
4. The gene expression system of claim 3, wherein the gene encoding green fluorescent protein in the linear plasmid is replaced with another gene of interest, said other gene of interest being a gene encoding a protein of interest.
5. Cells containing a linear plasmid;
the linear plasmid sequentially contains a left replication origin, a promoter, a target gene and a right replication origin, wherein the left replication origin is shown as SEQ ID No.3, and the right replication origin is shown as SEQ ID No. 4;
the cell also contains a plasmid carrying the gene cluster shown in SEQ ID NO.1, or the gene cluster shown in SEQ ID NO.1 is integrated on the genome of the cell;
the cell is a Bacillus subtilis cell.
6. The cell of claim 5, wherein the cell is Bacillus subtilis 168 or Bacillus subtilis 168 deltaspo0A::lox72；
The bacillus subtilis 168△spo0A::lox72Is a knockout in Bacillus subtilis 168spo0AA gene ofspo0AThe nucleotide sequence of the gene is shown in SEQ ID NO. 7.
7. A method for constructing the gene expression system according to any one of claims 1 to 4, comprising the steps of: (1) constructing a linear plasmid containing a left replication origin, a promoter, a target gene and a right replication origin; (2) transferring the linear plasmid constructed in the step (1) into a host cell integrated with a gene shown by SEQ ID NO.1, or transferring the linear plasmid constructed in the step (1) and a plasmid carrying the gene shown by SEQ ID NO.1 into the host cell.
8. The method according to claim 7, wherein the left replication origin, the promoter, the gene of interest and the right replication origin in step (1) are fused by Gibbson assembly.
9. Use of the gene expression system of any one of claims 1 to 4 for the expression of a natural protein, an artificial protein or a polypeptide.
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| 同源重组法构建枯草杆菌 spo0A 基因缺失突变株;余志强 等;《武汉大学学报(理学版)》;20040430;第50卷(第2期);第229页右栏最后一段，220页左栏第1段，标题 * |
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