CN114507685A - Bacillus subtilis polygene modular assembly and inducible expression plasmid and construction method thereof - Google Patents
Bacillus subtilis polygene modular assembly and inducible expression plasmid and construction method thereof Download PDFInfo
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Abstract
Based on the modular design concept of synthetic biology, the invention aims to provide a bacillus subtilis inducible expression plasmid (named pBsubPB01) containing a biological building block (BioBricks) module and a construction method thereof, wherein the plasmid can realize modular rapid assembly of a plurality of target genes and high-efficiency expression in bacillus subtilis, and relates to the technical fields of molecular biology, genetic engineering, synthetic biology and the like. The biological building block module constructed by the invention is as follows: isocaudarner (XbaI and NheI) are respectively placed at the upstream of a promoter and the downstream of a terminator of pHT01 (Escherichia coli-Bacillus subtilis shuttle plasmid), and an inducible chimeric promoter Pgrac, a ribosome binding site RBS, a multiple cloning site MCS and a prtA terminator are designed between two enzyme cutting sites. By utilizing the constructed biological building block module, a plurality of genes can be quickly connected in series in an operon form, and inducible coexpression in bacillus subtilis is realized. The plasmid is suitable for constructing a polygene synthesis way in the bacillus subtilis and has potential application value in synthetic biology with the bacillus subtilis as a chassis.
Description
Technical Field
The invention relates to the technical fields of molecular biology, genetic engineering, synthetic biology and the like, and particularly relates to a bacillus subtilis modular assembly based on a biological building block module, a construction method of a polygene expression plasmid and application thereof.
Background
The bacillus subtilis is a probiotic approved by the Ministry of agriculture in China, has no drug resistance to antibiotics, is nontoxic and harmless to people and livestock, is green and environment-friendly, has no pollution to the environment, is safe to crops, can generate various antibiotics, and has a good inhibiting effect on various animal, plant and human pathogenic bacteria. The bacteria also have strong lipase, protease and amylase activities, broad-spectrum antibacterial activity and strong anti-reverse capability. As a safe underpan cell, Bacillus subtilis is widely applied to the synthesis of various enzymes and compounds. However, there is currently a lack of a rapid and convenient tool for the coordinated co-expression of plasmids from multiple genes.
Biological building blocks (BioBricks) are an important content of synthetic biology, each block is provided with a standardized joint, and the blocks can be arbitrarily assembled into a higher-level system according to a certain operation flow. These standardized components and procedures form the main feature of synthetic biology that distinguishes synthetic biology from other disciplines of existing biology, namely "engineering". The design concept of synthetic biology and the nature of engineering are introduced into a heterologous expression system, so that the construction of a single expression system is simple and easy, and the improvement and multidirectional exploration of the expression system by researchers are facilitated.
Based on the standardized thought of synthetic biology, the biological building block module is introduced into a bacillus subtilis expression vector, so that modular rapid assembly of multiple target genes is realized, and the target genes are expressed in bacillus subtilis in a synergistic co-mode. The plasmid constructed by the invention is suitable for constructing and optimizing a synthetic route consisting of multiple genes and the like, and can meet the requirements of synthetic biology with bacillus subtilis as a chassis on such tools.
Disclosure of Invention
In view of the above, the present invention aims to provide a bacillus subtilis multigene modular assembly and inducible expression plasmid (named pBsubPB01) comprising biological building block modules and a construction method thereof, so as to realize rapid assembly, optimization and synergistic inducible expression of the multigenes in bacillus subtilis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus subtilis expression plasmid containing a BioBricks module and a construction method thereof, wherein the plasmid is closed circular double-stranded DNA.
Optionally, the BioBricks module comprises: isocaudarner (XbaI), inducible chimeric promoter Pgrac, ribosome binding site RBS, multiple cloning site MCS, prtA terminator, isocaudarner (NheI) and restriction endonuclease (BglII).
Optionally, the BioBricks module utilizes iterative use of isocaudarner to rapidly assemble a module containing a target gene, and the assembled target genes are connected in series in the form of an operon.
Optionally, the promoter is a bacillus subtilis inducible chimeric promoter Pgrac, which can be induced and expressed by isopropyl-beta-D-thiogalactoside (IPTG), and the terminator is a prtA terminator.
Optionally, the isocaudarner is XbaI and NheI.
Optionally, the plasmid also comprises ampicillin and chloramphenicol resistance genes, and is a shuttle plasmid of escherichia coli and bacillus subtilis.
The plasmid vector construction method comprises the following steps:
the plasmid constructed by the invention is based on a bacillus subtilis common plasmid pHT01, and in order to utilize the NheI and BglII enzyme cutting sites in a BioBricks module, the original NheI and BglII enzyme cutting sites in pHT01 need to be mutated. The specific operation is as follows:
performing PCR amplification by using pHT01 as a template and Frg 1F and Frg 1R as upstream and downstream primers (table 1), and naming an amplification product as Frg.1; PCR was performed using pHT01 as a template and Frg 2F and Frg 2R as upstream and downstream primers (Table 1), and the amplification product was designated as Frg.2. The reaction system of PCR amplification is as follows: 2X Phanta Max mix 25. mu.L (Biotech Co., Ltd., Nanjing Nuo Wei Zan), upstream and downstream primers (100. mu.M) each 0.5. mu.L, template DNA 1. mu.L, sterile ultrapure water 23. mu.L, total 50. mu.L. After the PCR reaction was completed, the remaining template DNA (pHT01 plasmid) was digested with the restriction enzyme DpnI (ThermoFisher scientific). The fragments of 4888-bp (Frg.1) and 3106-bp (Frg.2) were recovered by agarose gel electrophoresis, respectively. The recovered target fragment was subjected to one-step cloning (Nanjing Novozam Biotech Co., Ltd.) to obtain a recombinant plasmid pHT01_ NBXrmv in which the restriction sites NheI and BglII were eliminated.
A DNA sequence containing the BioBricks module (named BsbPB 01-Cassate) was synthesized by Shanghai Bioengineering Co., Ltd and cloned between KpnI and BglII sites of the vector pUC57 to give pUC 57-BsbPB 01-Cassate. Plasmids pHT01_ NBXrmv and pUC 57-BubPB 01-Cassate were subjected to double digestion with KpnI and BglII, respectively, and after electrophoresis in 1% agarose gel, 7707-bp and 261-bp DNA fragments were recovered, respectively. The target plasmid pBsubPB01 (plasmid structure shown in FIG. 1) containing the BioBricks module was obtained by ligation using T4 ligase (Biotech, Inc., Nuo Zan).
Compared with the prior art, the invention has the following beneficial effects:
the introduction of the biological building block (BioBricks) module into the Bacillus subtilis expression plasmid can realize the modular rapid assembly of multiple genes and the co-expression of the genes in the Bacillus subtilis. By utilizing the standardized and modularized structure, the operation process of expressing multiple genes in the bacillus subtilis is simplified. The inducible chimeric promoter Pgrac is used for controlling the expression of the target gene, so that the control of the expression time of the target gene is realized, and the inducible chimeric promoter Pgrac can be used for the expression of the target gene which is harmful to the chassis.
In the embodiment of the invention, the capability of pBsubPB01 to express exogenous genes is verified by using an EGFP (enhanced green fluorescent protein) coding gene and a LacZ (beta-galactosidase) coding gene of an enhanced green fluorescent protein; the modular rapid assembly, optimization and multi-gene synergistic co-expression capability of pBsubPB01 are verified through the assembly and expression of EGFP and LacZ with different copy numbers.
Drawings
FIG. 1 structural map of plasmid pBsubPB 01. Among them, the BioBricks module consists of isocaudarner (XbaI), Pgrac-inducible promoter, ribosome binding site RBS, multiple cloning site MCS, prtA terminator, isocaudarner (NheI) and restriction endonuclease (BglII).
FIG. 2 shows that the enhanced green fluorescent protein coding gene EGFP is used for verifying the foreign gene expression capacity of pBsubPB 01. The fluorescence signal is read by a multifunctional microplate reader, and the excitation light and the emission light are 488nm and 520nm respectively. Wherein, the bacillus subtilis containing a blank plasmid pBsubPB01 is used as a blank control, and the bacillus subtilis containing a recombinant plasmid pBsubPB01-EGFP is used as an experimental group.
FIG. 3. verifying the ability of pBsubPB01 to express foreign genes using the beta-galactosidase encoding gene LacZ. The picture shows the color development after adding substrate 5-bromo-4-chloro-3-indole-beta-D-galactoside (X-gal) to the fermentation broth and reacting for 30 minutes at 37 ℃. Wherein, the bacillus subtilis containing a blank plasmid pBsubPB01 is used as a blank control, and the bacillus subtilis containing a recombinant plasmid pBsubPB01-LacZ is used as an experimental group.
FIG. 4 is a wavelength scan of the product after digestion of X-gal with β -galactosidase. The substrate X-gal was digested with β -galactosidase at 37 ℃ for 30 minutes, the supernatant was taken, and the product was scanned for absorbance (230-1000nm) using a multifunctional microplate reader.
FIG. 5 quantitative analysis of the ability of pBsubPB01 to express foreign genes using beta-galactosidase. Adding substrate X-gal into the fermentation liquor, reacting at 37 deg.C for 30 min, and detecting the absorbance of the product at 660nm with multifunctional microplate reader (A)660). Wherein, the bacillus subtilis containing a blank plasmid pBsubPB01 is used as a blank control, and the bacillus subtilis containing a recombinant plasmid pBsubPB01-LacZ is used as an experimental group.
Fig. 6. modular assembly process and mechanism based on biobuilding blocks BioBricks. (a) Procedure for modular assembly of EGFP and LacZ using pBsubPB 01. (b) The restriction enzyme digestion and connection mechanism of XbaI and NheI. XbaI and NheI are isocaudarner, and the cohesive ends (both CTAG) after enzyme digestion are the same, so that the XbaI and the NheI can be connected with each other; but the ligated sequence is not cleaved by XbaI nor NheI, so that the next round of modular assembly is possible.
FIG. 7 plasmid constructs of pBsubPB01-EGFP-LacZ-LacZ and pBsubPB01-EGFP-LacZ-EGFP after BioBricks modular assembly. (a) Plasmid structure of pBsubPB01-EGFP-LacZ-LacZ after BioBricks modular assembly; (b) plasmid structure of pBsubPB01-EGFP-LacZ-EGFP after BioBricks modular assembly.
FIG. 8 quantitative analysis of Green fluorescence signals and beta-galactosidase Activity of strains expressing different copy numbers of EGFP and LacZ (A)660). Different copy numbers of EGFP and LacZ are modularly assembled by using pBsubPB01, and are co-expressed in Bacillus subtilis, and a green fluorescence signal and beta-galactosidase activity are respectively measured by using a multifunctional microplate reader.
Detailed description of the invention
The invention provides a plasmid for multigene modular assembly and inducible expression in bacillus subtilis based on a BioBricks module and a construction method thereof.
The plasmids and their use according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
The function of the constructed plasmid pBsubPB01 was verified by using the enhanced green fluorescent protein coding gene EGFP.
The plasmid pBsubPB01 was digested simultaneously with restriction enzymes BamHI and PstI, and the digested product was recovered. The EGFP gene was amplified using primer pairs BubPB 01-EGFP F and BubPB-EGFP R (Table 1), and the product was separated and purified by agarose gel electrophoresis and cloned into BamHI and PstI linearized pBsubPB01 using a one-step cloning kit (Nyjingo Biotech, Inc.) to give the recombinant plasmid pBsubPB 01-EGFP. The reaction system of PCR amplification is as follows: 2X Phanta Max mix 25. mu.L (Biotech Co., Ltd., Nanjing Nuo Zan), upstream and downstream primers (100. mu.M) each 0.5. mu.L, template DNA 1. mu.L, sterilized ultrapure water 23. mu.L, total 50. mu.L.
Plasmids pBsubPB01 and pBsubPB01-EGFP were transformed into Bacillus subtilis 168(Bacillus subtilis 168) strain, respectively, using chemical transformation. Culturing the recombinant bacillus subtilis in an LB culture medium at 37 ℃ for 2 hours at 200 rpm; IPTG was added to a final concentration of 1mM and incubation was continued under the same conditions for 8 hours. The cells were centrifuged at 4000rpm for 5min at 4 ℃ and were collected and resuspended in 100mM phosphate buffer (PBS, pH 7.0). And analyzing the green fluorescence signal by using a multifunctional microplate reader, and setting the wavelengths of the excitation light and the emission light to be 488nm and 520nm respectively. As a result, the fluorescence signal of the strain containing pBsubPB01-EGFP was 4.80X 104The fluorescence signal of the control group (strain containing empty pBsubPB01) was 0.41X 104The difference in fluorescence signal intensity was 11.71-fold (FIG. 2). The result proves that the constructed plasmid pBsubPB01 can efficiently express the exogenous gene EGFP in the bacillus subtilis.
TABLE 1 primers used in this patent
Example 2
The function of the constructed plasmid pBsubPB01 was verified using beta-galactosidase (encoded by LacZ).
The plasmid pBsubPB01 was digested simultaneously with restriction enzymes BamHI and PstI, and the digested product was recovered. The beta-galactosidase encoding gene LacZ was amplified using the primer pair BscubPB 01-LacZ (pGoal) F and BscubPB-LacZR, separated and purified by agarose gel electrophoresis, and cloned into BamHI and PstI linearized pBsubPB01 using a one-step cloning kit (Nyjingz Biotech, Inc.) to give the recombinant plasmid pBsubPB 01-LacZ. The reaction system of PCR amplification is as follows: 2X Phanta Max mix 25. mu.L (Biotech Co., Ltd., Nanjing Nuo Zan), upstream and downstream primers (100. mu.M) each 0.5. mu.L, template DNA 1. mu.L, sterilized ultrapure water 23. mu.L, total 50. mu.L.
Plasmids pBsubPB01 and pBsubPB01-LacZ were transformed into Bacillus subtilis 168 strain, respectively, using chemical transformation. Culturing in LB culture medium at 37 deg.C and 200rpm for 2 hr; IPTG was added to a final concentration of 1mM and incubation was continued under the same conditions for 8 hours. The cells were centrifuged at 4000rpm for 5min at 4 ℃ and were collected and resuspended in 100mM phosphate buffer (PBS, pH 7.0). 5-bromo-4-chloro-3-indole-. beta. -D-galactoside (X-gal) was added as a substrate to a final concentration of 0.4g/L, and incubated at 37 ℃ and 200rpm for 30 minutes to observe a color reaction. The results showed that the strain containing pBsubPB01-LacZ was able to hydrolyze the substrate X-gal to blue (with. beta. -galactosidase hydrolyzing activity), whereas the control (strain containing empty pBsubPB01) was unchanged in color (without. beta. -galactosidase hydrolyzing activity) (FIG. 3). The results demonstrate that the constructed plasmid pBsubPB01 can effectively express the foreign gene LacZ.
Example 3
The activity of beta-galactosidase was quantified by quantifying the hydrolysis product of X-gal.
Bacillus subtilis 168 containing plasmids pBsubPB01 and pBsubPB01-LacZ was cultured in LB medium at 37 ℃ and 200rpm for 2 hours, respectively, as in example 2; is added to a final concentration of1mM IPTG, and incubation was continued under the same conditions for 8 hours. The cells were centrifuged at 4000rpm for 5min at 4 ℃ and were collected and resuspended in 100mM phosphate buffer (PBS, pH 7.0). X-gal was added as a substrate to a final concentration of 0.4g/L, and incubated at 37 ℃ for 30 minutes at 200 rpm. Centrifuging at 12000rpm for 2min, and collecting supernatant. The supernatant containing the beta-galactosidase expressed by the plasmid pBsubPB01-LacZ was used for the absorption wavelength scan (230-1000 nm). The results show that the hydrolysis product of X-gal has a maximum absorption peak at 660nm (FIG. 4). Therefore, absorbance at 660nm (A)660) Can be used for the quantitative analysis of X-gal hydrolysate, namely the quantitative analysis of beta-galactosidase activity. The absorbance at 660nm of the fermentation supernatants of the strains containing pBsubPB01-LacZ and pBsubPB01 (A) was determined separately660). As a result, it was found that A of the strain containing pBsubPB01-LacZ6601.11, and A of the control group (pBsubPB 01-containing strain)6600.01, which differs by a factor of 111 (FIG. 5). The result proves that the constructed plasmid pBsubPB01 can efficiently express the foreign gene LacZ in the bacillus subtilis.
Example 4
The constructed plasmid pBsubPB01 was used to modularly assemble a foreign gene and co-express it in Bacillus subtilis.
To verify the modular rapid assembly capability of the constructed plasmids, modular assembly was performed using EGFP and LacZ. The specific operation is as follows: cutting pBsubPB01-EGFP by NheI and BglII, separating by agarose gel electrophoresis and recovering a 8663-bp fragment (containing pBsubPB01 plasmid skeleton and EGFP gene); pBsubPB01-LacZ was digested with XbaI and BglII, and the 3357-bp fragment (containing the Pgrac promoter-LacZ gene-prtA terminator structure) was separated and recovered by agarose gel electrophoresis. The recovered fragments were ligated using T4 ligase to obtain recombinant plasmid pBsubPB01-EGFP-LacZ in tandem with an operon structure (see FIG. 6a for a process).
Since XbaI and NheI are isocaudards, they have the same cohesive end CTAG and can therefore be religated; however, since the ligated sequence differs from XbaI and NheI, it cannot be cleaved by XbaI or NheI (FIG. 6b), and the resulting recombinant plasmid pBsubPB01-EGFP-LacZ can be subjected to the next round of assembly by the same strategy as above using a combination of the isocaudarymes (XbaI and NheI) and BglII. The specific operation is as follows: the enzyme pBsubPB01-EGFP-LacZ was digested with NheI and BglII, and the 12008-bp fragment (containing pBsubPB01 plasmid backbone, EGFP and LacZ genes) was separated and recovered by agarose gel electrophoresis; pBsubPB01-LacZ was digested with XbaI and BglII, and the 3357-bp fragment (containing the Pgram promoter-LacZ gene-prtA terminator structure) was separated and recovered by agarose gel electrophoresis. The recovered fragments were ligated using T4 ligase to obtain recombinant plasmid pBsubPB01-EGFP-LacZ-LacZ (containing 1 copy of EGFP and 2 copies of LacZ, plasmid structure shown in FIG. 7a) in tandem with an operon structure. The enzyme pBsubPB01-EGFP-LacZ was digested with NheI and BglII, and the 12008-bp fragment (containing pBsubPB01 plasmid backbone, EGFP and LacZ genes) was separated and recovered by agarose gel electrophoresis; pBsubPB01-EGFP was digested with XbaI and BglII, and a 966-bp fragment (containing the Pgrac promoter-EGFP gene-prtA terminator structure) was separated and recovered by agarose gel electrophoresis. The recovered fragments were ligated using T4 ligase to obtain recombinant plasmid pBsubPB01-EGFP-LacZ-EGFP (containing 2 copies of EGFP and 1 copy of LacZ, plasmid structure shown in FIG. 7b) in tandem with an operon structure.
As in example 2 and example 3, recombinant plasmids pBsubPB01-EGFP-LacZ, pBsubPB01-EGFP-LacZ-LacZ and pBsubPB01-EGFP-LacZ-EGFP were transformed into Bacillus subtilis 168, respectively, and after culture and IPTG induction, the cells were collected and resuspended in phosphate buffer. The recombinant cells were assayed for green fluorescence signal (as in example 2) and β -galactosidase activity (as in example 3), respectively. The results show that after two exogenous genes EGFP and LacZ are co-expressed, the same strain has both green fluorescence signals and beta-galactosidase activity (FIG. 8). The result proves that the constructed plasmid can realize the co-expression of 2 or more exogenous genes. The constructed plasmids are used for modular assembly to rapidly increase the copy number of the gene, and the result shows that when the copy numbers of EGFP and LacZ are both 1 (containing the plasmid pBsubPB01-EGFP-LacZ), the green fluorescence signal is 4.63 multiplied by 104，A660(β -galactosidase activity) 1.01; when the copy number of EGFP and LacZ was increased to 2, the green fluorescence signal was 7.04×104(containing plasmid pBsubPB01-EGFP-LacZ-EGFP), A660(beta-galactosidase activity) increased to 1.71 (containing plasmid pBsubPB01-EGFP-LacZ-LacZ), 52.1% and 69.3%, respectively (FIG. 8). The result proves that the constructed plasmid can express a plurality of exogenous genes in the bacillus subtilis through modular rapid assembly.
The foregoing is only an alternative embodiment of the present invention, and it should be noted that modifications and embellishments could be made by those skilled in the art without departing from the principle of the present invention, and these should be considered as the protection scope of the present invention.
Sequence listing
<110> Luyongkun
Xu Jingliang
Ren Jing
Zhang Hui
Zhu Lijuan
Xiong Wenlong
Allammu (Md. Asraful Alam)
Qu Lingbo
Ying Hanjie
Wang Shiyuan
Field with remained height
<120> bacillus subtilis polygene modular assembly and inducible expression plasmid and construction method thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 271
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
ggtacctcta gaagctattg taacataatc ggtacggggg tgaaaaagct aacggaaaag 60
ggagcggaaa agaatgatgt aagcgtgaaa aattttttat cttatcactt gaaattggaa 120
gggagattct ttattataag aattgtggaa ttgtgagcgg ataacaattc ccaattaaag 180
gaggaaggat ccctgcaggt cgacgtcccc ggggcagccc gcctaatgag cgggcttttt 240
tcacgtcacg cgtgctagcc catggagatc t 271
<210> 2
<211> 153
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
gaaaagaatg atgtaagcgt gaaaaatttt ttatcttatc acttgaaatt ggaagggaga 60
ttctttatta taagaattgt ggaattgtga gcggataaca attcccaatt aaaggaggaa 120
ggatccctgc aggtcgacgt ccccggggca gcc 153
<210> 3
<211> 8
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
aaaggagg 8
<210> 4
<211> 27
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
ggatccctgc aggtcgacgt ccccggg 27
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 5
agcccgccta atgagcgggc tttt 24
<210> 6
<211> 717
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 6
atgggtaagg gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaagcttc ctgttccttg gccaacactt 180
gtcactactc ttacttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 240
cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc 300
aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 360
aacagaatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 420
ttggaataca actacaactc ccacaacgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717
<210> 7
<211> 3108
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 7
atgcgcccaa acatgcatgg atgcgttgag atgaggatga gggaagcaag aatggatccc 60
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 120
gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 180
caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg 240
gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca 300
aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg 360
gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat 420
gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttaactcg 480
gcgtttcatc tgtggtgcaa cgggcgctgg gtcggttacg gccaggacag tcgtttgccg 540
tctgaatttg acctgagcgc atttttacgc gccggagaaa accgcctcgc ggtgatggtg 600
ctgcgttgga gtgacggcag ttatctggaa gatcaggata tgtggcggat gagcggcatt 660
ttccgtgacg tctcgttgct gcataaaccg actacacaaa tcagcgattt ccatgttgcc 720
actcgcttta atgatgattt cagccgcgct gtactggagg ctgaagttca gatgtgcggc 780
gagttgcgtg actacctacg ggtaacagtt tctttatggc agggtgaaac gcaggtcgcc 840
agcggcaccg cgcctttcgg cggtgaaatt atcgatgagc gtggtggtta tgccgatcgc 900
gtcacactac gtctgaacgt cgaaaacccg aaactgtgga gcgccgaaat cccgaatctc 960
tatcgtgcgg tggttgaact gcacaccgcc gacggcacgc tgattgaagc agaagcctgc 1020
gatgtcggtt tccgcgaggt gcggattgaa aatggtctgc tgctgctgaa cggcaagccg 1080
ttgctgattc gaggcgttaa ccgtcacgag catcatcctc tgcatggtca ggtcatggat 1140
gagcagacga tggtgcagga tatcctgctg atgaagcaga acaactttaa cgccgtgcgc 1200
tgttcgcatt atccgaacca tccgctgtgg tacacgctgt gcgaccgcta cggcctgtat 1260
gtggtggatg aagccaatat tgaaacccac ggcatggtgc caatgaatcg tctgaccgat 1320
gatccgcgct ggctaccggc gatgagcgaa cgcgtaacgc gaatggtgca gcgcgatcgt 1380
aatcacccga gtgtgatcat ctggtcgctg gggaatgaat caggccacgg cgctaatcac 1440
gacgcgctgt atcgctggat caaatctgtc gatccttccc gcccggtgca gtatgaaggc 1500
ggcggagccg acaccacggc caccgatatt atttgcccga tgtacgcgcg cgtggatgaa 1560
gaccagccct tcccggctgt gccgaaatgg tccatcaaaa aatggctttc gctacctgga 1620
gagacgcgcc cgctgatcct ttgcgaatac gcccacgcga tgggtaacag tcttggcggt 1680
ttcgctaaat actggcaggc gtttcgtcag tatccccgtt tacagggcgg cttcgtctgg 1740
gactgggtgg atcagtcgct gattaaatat gatgaaaacg gcaacccgtg gtcggcttac 1800
ggcggtgatt ttggcgatac gccgaacgat cgccagttct gtatgaacgg tctggtcttt 1860
gccgaccgca cgccgcatcc agcgctgacg gaagcaaaac accagcagca gtttttccag 1920
ttccgtttat ccgggcaaac catcgaagtg accagcgaat acctgttccg tcatagcgat 1980
aacgagctcc tgcactggat ggtggcgctg gatggtaagc cgctggcaag cggtgaagtg 2040
cctctggatg tcgctccaca aggtaaacag ttgattgaac tgcctgaact accgcagccg 2100
gagagcgccg ggcaactctg gctcacagta cgcgtagtgc aaccgaacgc gaccgcatgg 2160
tcagaagccg ggcacatcag cgcctggcag cagtggcgtc tggcggaaaa cctcagtgtg 2220
acgctccccg ccgcgtccca cgccatcccg catctgacca ccagcgaaat ggatttttgc 2280
atcgagctgg gtaataagcg ttggcaattt aaccgccagt caggctttct ttcacagatg 2340
tggattggcg ataaaaaaca actgctgacg ccgctgcgcg atcagttcac ccgtgcaccg 2400
ctggataacg acattggcgt aagtgaagcg acccgcattg accctaacgc ctgggtcgaa 2460
cgctggaagg cggcgggcca ttaccaggcc gaagcagcgt tgttgcagtg cacggcagat 2520
acacttgctg atgcggtgct gattacgacc gctcacgcgt ggcagcatca ggggaaaacc 2580
ttatttatca gccggaaaac ctaccggatt gatggtagtg gtcaaatggc gattaccgtt 2640
gatgttgaag tggcgagcga tacaccgcat ccggcgcgga ttggcctgaa ctgccagctg 2700
gcgcaggtag cagagcgggt aaactggctc ggattagggc cgcaagaaaa ctatcccgac 2760
cgccttactg ccgcctgttt tgaccgctgg gatctgccat tgtcagacat gtataccccg 2820
tacgtcttcc cgagcgaaaa cggtctgcgc tgcgggacgc gcgaattgaa ttatggccca 2880
caccagtggc gcggcgactt ccagttcaac atcagccgct acagtcaaca gcaactgatg 2940
gaaaccagcc atcgccatct gctgcacgcg gaagaaggca catggctgaa tatcgacggt 3000
ttccatatgg ggattggtgg cgacgactcc tggagcccgt cagtatcggc ggaattccag 3060
ctgagcgccg gtcgctacca ttaccagttg gtctggtgtc aaaaataa 3108
<210> 8
<211> 7956
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 8
ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60
tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120
gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180
aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240
tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300
ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360
gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420
ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480
tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540
cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600
aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660
tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720
cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780
tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840
ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900
ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960
aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020
ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080
tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1140
ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1200
aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1260
cgagctcagg ccttaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 1320
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1380
tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 1440
tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 1500
gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 1560
cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 1620
ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 1680
tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 1740
ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 1800
ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 1860
gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 1920
tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 1980
tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 2040
gattgtgcac cgccgtttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 2100
cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 2160
gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 2220
gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 2280
ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 2340
cggcatactc tgcgacatcg tataacgtta ctggtttcat caaaatcgtc tccctccgtt 2400
tgaatatttg attgatcgta accagatgaa gcactctttc cactatccct acagtgttat 2460
ggcttgaaca atcacgaaac aataattggt acgtacgatc tttcagccga ctcaaacatc 2520
aaatcttaca aatgtagtct ttgaaagtat tacatatgta agatttaaat gcaaccgttt 2580
tttcggaagg aaatgatgac ctcgtttcca ccggaattag cttggtacca gctattgtaa 2640
cataatcggt acgggggtga aaaagctaac ggaaaaggga gcggaaaaga atgatgtaag 2700
cgtgaaaaat tttttatctt atcacttgaa attggaaggg agattcttta ttataagaat 2760
tgtggaattg tgagcggata acaattccca attaaaggag gaaggatcct ctagagtcga 2820
cgtccccggg gcagcccgcc taatgagcgg gcttttttca cgtcacgcgt ccatggagat 2880
ctttgtctgc aactgaaaag tttatacctt acctggaaca aatggttgaa acatacgagg 2940
ctaatatcgg cttattagga atagtccctg tactaataaa atcaggtgga tcagttgatc 3000
agtatatttt ggacgaagct cggaaagaat ttggagatga cttgcttaat tccacaatta 3060
aattaaggga aagaataaag cgatttgatg ttcaaggaat cacggaagaa gatactcatg 3120
ataaagaagc tctaaaacta ttcaataacc ttacaatgga attgatcgaa agggtggaag 3180
gttaatggta cgaaaattag gggatctacc tagaaagcca caaggcgata ggtcaagctt 3240
aaagaaccct tacatggatc ttacagattc tgaaagtaaa gaaacaacag aggttaaaca 3300
aacagaacca aaaagaaaaa aagcattgtt gaaaacaatg aaagttgatg tttcaatcca 3360
taataagatt aaatcgctgc acgaaattct ggcagcatcc gaagggaatt catattactt 3420
agaggatact attgagagag ctattgataa gatggttgag acattacctg agagccaaaa 3480
aactttttat gaatatgaat taaaaaaaag aaccaacaaa ggctgagaca gactccaaac 3540
gagtctgttt ttttaaaaaa aatattagga gcattgaata tatattagag aattaagaaa 3600
gacatgggaa taaaaatatt ttaaatccag taaaaatatg ataagattat ttcagaatat 3660
gaagaactct gtttgttttt gatgaaaaaa caaacaaaaa aaatccacct aacggaatct 3720
caatttaact aacagcggcc aaactgagaa gttaaatttg agaaggggaa aaggcggatt 3780
tatacttgta tttaactatc tccattttaa cattttatta aaccccatac aagtgaaaat 3840
cctcttttac actgttcctt taggtgatcg cggagggaca ttatgagtga agtaaaccta 3900
aaaggaaata cagatgaatt agtgtattat cgacagcaaa ccactggaaa taaaatcgcc 3960
aggaagagaa tcaaaaaagg gaaagaagaa gtttattatg ttgctgaaac ggaagagaag 4020
atatggacag aagagcaaat aaaaaacttt tctttagaca aatttggtac gcatatacct 4080
tacatagaag gtcattatac aatcttaaat aattacttct ttgatttttg gggctatttt 4140
ttaggtgctg aaggaattgc gctctatgct cacctaactc gttatgcata cggcagcaaa 4200
gacttttgct ttcctagtct acaaacaatc gctaaaaaaa tggacaagac tcctgttaca 4260
gttagaggct acttgaaact gcttgaaagg tacggtttta tttggaaggt aaacgtccgt 4320
aataaaacca aggataacac agaggaatcc ccgattttta agattagacg taaggttcct 4380
ttgctttcag aagaactttt aaatggaaac cctaatattg aaattccaga tgacgaggaa 4440
gcacatgtaa agaaggcttt aaaaaaggaa aaagagggtc ttccaaaggt tttgaaaaaa 4500
gagcacgatg aatttgttaa aaaaatgatg gatgagtcag aaacaattaa tattccagag 4560
gccttacaat atgacacaat gtatgaagat atactcagta aaggagaaat tcgaaaagaa 4620
atcaaaaaac aaatacctaa tcctacaaca tcttttgaga gtatatcaat gacaactgaa 4680
gaggaaaaag tcgacagtac tttaaaaagc gaaatgcaaa atcgtgtctc taagccttct 4740
tttgatacct ggtttaaaaa cactaagatc aaaattgaaa ataaaaattg tttattactt 4800
gtaccgagtg aatttgcatt tgaatggatt aagaaaagat atttagaaac aattaaaaca 4860
gtccttgaag aagctggata tgttttcgaa aaaatcgaac taagaaaagt gcaataaact 4920
gctgaagtat ttcagcagtt ttttttattt agaaatagtg aaaaaaatat aatcagggag 4980
gtatcaatat ttaatgagta ctgatttaaa tttatttaga ctggaattaa taattaacac 5040
gtagactaat taaaatttaa tgagggataa agaggataca aaaatattaa tttcaatccc 5100
tattaaattt taacaagggg gggattaaaa tttaattaga ggtttatcca caagaaaaga 5160
ccctaataaa atttttacta gggttataac actgattaat ttcttaatgg gggagggatt 5220
aaaatttaat gacaaagaaa acaatctttt aagaaaagct tttaaaagat aataataaaa 5280
agagctttgc gattaagcaa aactctttac tttttcattg acattatcaa attcatcgat 5340
ttcaaattgt tgttgtatca taaagttaat tctgttttgc acaacctttt caggaatata 5400
aaacacatct gaggcttgtt ttataaactc agggtcgcta aagtcaatgt aacgtagcat 5460
atgatatggt atagcttcca cccaagttag cctttctgct tcttctgaat gtttttcata 5520
tacttccatg ggtatctcta aatgattttc ctcatgtagc aaggtatgag caaaaagttt 5580
atggaattga tagttcctct ctttttcttc aactttttta tctaaaacaa acactttaac 5640
atctgagtca atgtaagcat aagatgtttt tccagtcata atttcaatcc caaatctttt 5700
agacagaaat tctggacgta aatcttttgg tgaaagaatt tttttatgta gcaatatatc 5760
cgatacagca ccttctaaaa gcgttggtga atagggcatt ttacctatct cctctcattt 5820
tgtggaataa aaatagtcat attcgtccat ctacctatcc tattatcgaa cagttgaact 5880
ttttaatcaa ggatcagtcc tttttttcat tattcttaaa ctgtgctctt aactttaaca 5940
actcgatttg tttttccaga tctcgagggt aactagcctc gccgatcccg caagaggccc 6000
ggcagtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 6060
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 6120
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 6180
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 6240
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 6300
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6360
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 6420
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 6480
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 6540
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 6600
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 6660
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 6720
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 6780
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 6840
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 6900
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6960
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 7020
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 7080
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 7140
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 7200
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 7260
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 7320
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7380
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7440
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 7500
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 7560
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 7620
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 7680
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 7740
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 7800
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 7860
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 7920
tgagcgagga agcggaagag cgcccaatac gcatgc 7956
<210> 9
<211> 7968
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 9
ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60
tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120
gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180
aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240
tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300
ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360
gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420
ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480
tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540
cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600
aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660
tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720
cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780
tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840
ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900
ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960
aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020
ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080
tatggcgtgc tggtagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1140
ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1200
aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1260
cgagctcagg ccttaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 1320
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1380
tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 1440
tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 1500
gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 1560
cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 1620
ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 1680
tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 1740
ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 1800
ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 1860
gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 1920
tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 1980
tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 2040
gattgtgcac cgccgtttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 2100
cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 2160
gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 2220
gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 2280
ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 2340
cggcatactc tgcgacatcg tataacgtta ctggtttcat caaaatcgtc tccctccgtt 2400
tgaatatttg attgatcgta accagatgaa gcactctttc cactatccct acagtgttat 2460
ggcttgaaca atcacgaaac aataattggt acgtacgatc tttcagccga ctcaaacatc 2520
aaatcttaca aatgtagtct ttgaaagtat tacatatgta agatttaaat gcaaccgttt 2580
tttcggaagg aaatgatgac ctcgtttcca ccggaattag cttggtacct ctagaagcta 2640
ttgtaacata atcggtacgg gggtgaaaaa gctaacggaa aagggagcgg aaaagaatga 2700
tgtaagcgtg aaaaattttt tatcttatca cttgaaattg gaagggagat tctttattat 2760
aagaattgtg gaattgtgag cggataacaa ttcccaatta aaggaggaag gatccctgca 2820
ggtcgacgtc cccggggcag cccgcctaat gagcgggctt ttttcacgtc acgcgtgcta 2880
gcccatggag atctttgtct gcaactgaaa agtttatacc ttacctggaa caaatggttg 2940
aaacatacga ggctaatatc ggcttattag gaatagtccc tgtactaata aaatcaggtg 3000
gatcagttga tcagtatatt ttggacgaag ctcggaaaga atttggagat gacttgctta 3060
attccacaat taaattaagg gaaagaataa agcgatttga tgttcaagga atcacggaag 3120
aagatactca tgataaagaa gctctaaaac tattcaataa ccttacaatg gaattgatcg 3180
aaagggtgga aggttaatgg tacgaaaatt aggggatcta cctagaaagc cacaaggcga 3240
taggtcaagc ttaaagaacc cttacatgga tcttacagat tctgaaagta aagaaacaac 3300
agaggttaaa caaacagaac caaaaagaaa aaaagcattg ttgaaaacaa tgaaagttga 3360
tgtttcaatc cataataaga ttaaatcgct gcacgaaatt ctggcagcat ccgaagggaa 3420
ttcatattac ttagaggata ctattgagag agctattgat aagatggttg agacattacc 3480
tgagagccaa aaaacttttt atgaatatga attaaaaaaa agaaccaaca aaggctgaga 3540
cagactccaa acgagtctgt ttttttaaaa aaaatattag gagcattgaa tatatattag 3600
agaattaaga aagacatggg aataaaaata ttttaaatcc agtaaaaata tgataagatt 3660
atttcagaat atgaagaact ctgtttgttt ttgatgaaaa aacaaacaaa aaaaatccac 3720
ctaacggaat ctcaatttaa ctaacagcgg ccaaactgag aagttaaatt tgagaagggg 3780
aaaaggcgga tttatacttg tatttaacta tctccatttt aacattttat taaaccccat 3840
acaagtgaaa atcctctttt acactgttcc tttaggtgat cgcggaggga cattatgagt 3900
gaagtaaacc taaaaggaaa tacagatgaa ttagtgtatt atcgacagca aaccactgga 3960
aataaaatcg ccaggaagag aatcaaaaaa gggaaagaag aagtttatta tgttgctgaa 4020
acggaagaga agatatggac agaagagcaa ataaaaaact tttctttaga caaatttggt 4080
acgcatatac cttacataga aggtcattat acaatcttaa ataattactt ctttgatttt 4140
tggggctatt ttttaggtgc tgaaggaatt gcgctctatg ctcacctaac tcgttatgca 4200
tacggcagca aagacttttg ctttcctagt ctacaaacaa tcgctaaaaa aatggacaag 4260
actcctgtta cagttagagg ctacttgaaa ctgcttgaaa ggtacggttt tatttggaag 4320
gtaaacgtcc gtaataaaac caaggataac acagaggaat ccccgatttt taagattaga 4380
cgtaaggttc ctttgctttc agaagaactt ttaaatggaa accctaatat tgaaattcca 4440
gatgacgagg aagcacatgt aaagaaggct ttaaaaaagg aaaaagaggg tcttccaaag 4500
gttttgaaaa aagagcacga tgaatttgtt aaaaaaatga tggatgagtc agaaacaatt 4560
aatattccag aggccttaca atatgacaca atgtatgaag atatactcag taaaggagaa 4620
attcgaaaag aaatcaaaaa acaaatacct aatcctacaa catcttttga gagtatatca 4680
atgacaactg aagaggaaaa agtcgacagt actttaaaaa gcgaaatgca aaatcgtgtc 4740
tctaagcctt cttttgatac ctggtttaaa aacactaaga tcaaaattga aaataaaaat 4800
tgtttattac ttgtaccgag tgaatttgca tttgaatgga ttaagaaaag atatttagaa 4860
acaattaaaa cagtccttga agaagctgga tatgttttcg aaaaaatcga actaagaaaa 4920
gtgcaataaa ctgctgaagt atttcagcag ttttttttat ttagaaatag tgaaaaaaat 4980
ataatcaggg aggtatcaat atttaatgag tactgattta aatttattta gactggaatt 5040
aataattaac acgtagacta attaaaattt aatgagggat aaagaggata caaaaatatt 5100
aatttcaatc cctattaaat tttaacaagg gggggattaa aatttaatta gaggtttatc 5160
cacaagaaaa gaccctaata aaatttttac tagggttata acactgatta atttcttaat 5220
gggggaggga ttaaaattta atgacaaaga aaacaatctt ttaagaaaag cttttaaaag 5280
ataataataa aaagagcttt gcgattaagc aaaactcttt actttttcat tgacattatc 5340
aaattcatcg atttcaaatt gttgttgtat cataaagtta attctgtttt gcacaacctt 5400
ttcaggaata taaaacacat ctgaggcttg ttttataaac tcagggtcgc taaagtcaat 5460
gtaacgtagc atatgatatg gtatagcttc cacccaagtt agcctttctg cttcttctga 5520
atgtttttca tatacttcca tgggtatctc taaatgattt tcctcatgta gcaaggtatg 5580
agcaaaaagt ttatggaatt gatagttcct ctctttttct tcaacttttt tatctaaaac 5640
aaacacttta acatctgagt caatgtaagc ataagatgtt tttccagtca taatttcaat 5700
cccaaatctt ttagacagaa attctggacg taaatctttt ggtgaaagaa tttttttatg 5760
tagcaatata tccgatacag caccttctaa aagcgttggt gaatagggca ttttacctat 5820
ctcctctcat tttgtggaat aaaaatagtc atattcgtcc atctacctat cctattatcg 5880
aacagttgaa ctttttaatc aaggatcagt cctttttttc attattctta aactgtgctc 5940
ttaactttaa caactcgatt tgtttttcca gatgtcgagg gtaactagcc tcgccgatcc 6000
cgcaagaggc ccggcagtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 6060
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 6120
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 6180
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 6240
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 6300
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 6360
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 6420
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 6480
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 6540
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 6600
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 6660
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 6720
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 6780
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 6840
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 6900
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 6960
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 7020
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 7080
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 7140
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 7200
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 7260
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 7320
atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 7380
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 7440
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 7500
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 7560
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 7620
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 7680
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 7740
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 7800
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 7860
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 7920
gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcatgc 7968
Claims (7)
1. A bacillus subtilis induced expression plasmid containing a biological building block (BioBricks) module and a construction method thereof are characterized in that the plasmid can rapidly assemble a plurality of target genes by utilizing the BioBricks module so as to realize the co-expression of the genes in bacillus subtilis.
2. The plasmid of claim 1, wherein the BioBricks module modularly assembles a plurality of genes of interest using iterative use of isocaudards.
3. The plasmid according to claim 1, characterized in that the BioBricks module (in 5 '→ 3' order) consists of a isocaudarner (XbaI), a promoter, a ribosome binding site RBS, a multiple cloning site MCS, a terminator, an isocaudarner (NheI) and a restriction endonuclease (BglII).
4. The plasmid of claim 1 and claim 3, wherein the promoter is a Bacillus subtilis inducible chimeric promoter Pgrac, which is inducible by isopropyl- β -D-thiogalactoside (IPTG), and the terminator is a Bacillus subtilis high efficiency terminator prtA.
5. The plasmid of claim 1, wherein the plasmid is a shuttle plasmid constructed, amplified and stored in E.coli and used for expressing a gene of interest in Bacillus subtilis.
6. The plasmid of claim 1, wherein the gene of interest is a gene encoding any protein desired to be expressed in Bacillus subtilis.
7. The plasmid of claim 1 to claim 5, wherein the plasmid can be used for modular assembly of genes of interest by means of a biobuilding block (BioBricks) module, theoretically allowing unlimited assembly of genes encoding desired expressed proteins within the load bearing range.
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