CN104073509B - A kind of method improving escherichia coli alcohol resistance - Google Patents
A kind of method improving escherichia coli alcohol resistance Download PDFInfo
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- CN104073509B CN104073509B CN201410311102.5A CN201410311102A CN104073509B CN 104073509 B CN104073509 B CN 104073509B CN 201410311102 A CN201410311102 A CN 201410311102A CN 104073509 B CN104073509 B CN 104073509B
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 28
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- 238000000034 method Methods 0.000 title claims abstract description 15
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- 101100327358 Rattus norvegicus Cdk12 gene Proteins 0.000 claims abstract description 11
- 239000013612 plasmid Substances 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 15
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- 230000001580 bacterial effect Effects 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 12
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 8
- 102000040945 Transcription factor Human genes 0.000 description 4
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- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
A kind of method improving escherichia coli alcohol resistance of disclosure, comprise the following steps: by the wild type fnr gene clone in coli strain to pKSC plasmid, obtain recombinant vector pKSC-fnr, replaced by base, obtain recombinant vector pKSC-fnr1 and the pKSC-fnr2 optimized；Finally replace with fnr1 and the fnr2 gene in pKSC-fnr1 and pKSC-fnr2<i>e. coli</i>wild type fnr gene in DH5 α, obtains the bacterial strain that two alcohol resistances improve.The present invention, by the base of the wild type fnr gene in coli strain is replaced, affects its gene relevant to alcohol resistance, and then improves the alcohol resistance in escherichia coli.The method can be effectively improved colibacillary alcohol resistance, has great importance for industrial production of ethyl alcohol to utilizing escherichia coli.
Description
Technical field
The invention belongs to biological chemical field, particularly to a kind of method improving escherichia coli alcohol resistance.
Background technology
Escherichia coli have that growth is fast, technical operation is relatively easy, large scale fermentation low cost and other advantages, are the type strain in current genetic engineering and the most commonly used foreign protein prokaryotic expression system.This system can produce the metabolite that many people need as " cell factory ", but thalline usually needs to cultivate in some hostile environments, the accumulation of various metabolites, not only can reduce the aggregate velocity of thalline self RNA, DNA, albumen and lipid etc., and seriously suppress the growth of recombinant bacterium.Thalline is effectively transformed, improves escherichia coli patience and adaptability to adverse environment condition and become the required major issue solved of microbial technique application.Thalline is to made self the regulating of environment change, or some mal-condition patiences and adaptive formation relate to the adjustment of whole metabolism network, and by many gene combineds effect, its Related Mechanism is still without completely clearly at present.
Transcription factor is a kind of protein molecule with special construction, enforcement controlling gene expressive function, and it controls the switch of genetic transcription, strengthens or weaken.Most cells function is interacted to form functional module by one group of gene and jointly performs, when the number gene of transcription factor regulation and control is very big, it participates in the probability of various kinds of cell function and is also greatly increased, it means that transcription factor is relevant to the function diversification being adjusted gene.Transcription factor FNR is just being in the superiors of gene transcription regulation network, it is possible to the transcriptional level of direct or indirect regulation and control thalline full-length genome, it is possible to set up certain contact between FNR and various kinds of cell function.Therefore, it can the protein structure by changing FNR, affect its transcriptional level being regulated and controled various gene, regulate the metabolism network of whole thalline, and then improve colibacillary thalline character.
Summary of the invention
It is an object of the invention to the shortcoming overcoming prior art with not enough, it is provided that a kind of method improving escherichia coli alcohol resistance.
It is an object of the invention to be achieved through the following technical solutions: a kind of method improving escherichia coli alcohol resistance, comprise the following steps:
(1) by coli strainE.coliWild type fnr gene clone in DH5 α is to pKSC plasmid, obtain recombinant vector pKSC-fnr, the sequence of wild type fnr gene is such as shown in SEQIDNO.1, the aminoacid sequence of wild type fnr is such as shown in SEQIDNO.2, the sequence of pKSC plasmid is such as shown in SEQIDNO.3, and the sequence of recombinant vector pKSC-fnr is such as shown in SEQIDNO.4；
(2) base is replaced: the fnr gene on recombinant vector pKSC-fnr obtain step (1) carries out base replacement: G164A, C182T, obtains the recombinant vector pKSC-fnr1 optimized；Base is replaced: C409G, G665C, obtains the recombinant vector pKSC-fnr2 optimized；The sequence of fnr1 gene is such as shown in SEQIDNO.5, the aminoacid sequence of fnr1 is such as shown in SEQIDNO.6, the sequence of fnr2 gene is such as shown in SEQIDNO.7, the aminoacid sequence of fnr2 is such as shown in SEQIDNO.8, the sequence of pKSC-fnr1 is such as shown in SEQIDNO.9, and the sequence of pKSC-fnr2 is such as shown in SEQIDNO.10；
(3) coli strain is replaced with fnr1 and the fnr2 gene in pKSC-fnr1 and pKSC-fnr2E.coliWild type fnr gene in DH5 α, obtains the coli strain that alcohol resistance improvesE.coliDH5 α fnr::fnr1 andE.coliDH5αfnr::fnr2。
The invention has the beneficial effects as follows: by the base of the wild type fnr gene in coli strain is replaced, affect the transcriptional level of its various genes regulated and controled, regulate the metabolism network of whole thalline, and then improve colibacillary alcohol resistance, have great importance for industrial production of ethyl alcohol to utilizing escherichia coli.
Accompanying drawing explanation
Fig. 1: recombinant vector pKSC-fnr structural representation；
Fig. 2: bacterial strain of the present inventionE.coliDH5 α fnr::fnr1 andE.coliDH5 α fnr::fnr2 growing state figure in 50g/l ethanol.
Detailed description of the invention
DNA sequence and the aminoacid sequence of this wild type fnr provide respectively in SEQIDNO.1 and SEQIDNO.2.
A kind of method improving escherichia coli alcohol resistance of the present invention, comprises the following steps:
1, by coli strainE.coliWild type fnr gene clone in DH5 α is to pKSC plasmid, obtain recombinant vector pKSC-fnr, the sequence of wild type fnr gene is such as shown in SEQIDNO.1, the aminoacid sequence of wild type fnr is such as shown in SEQIDNO.2, the sequence of pKSC plasmid is such as shown in SEQIDNO.3, and the sequence of recombinant vector pKSC-fnr is such as shown in SEQIDNO.4；
2, base is replaced: the fnr gene on recombinant vector pKSC-fnr obtain step (1) carries out base replacement: G164A, C182T, obtains the recombinant vector pKSC-fnr1 optimized；Base is replaced: C409G, G665C, obtains the recombinant vector pKSC-fnr2 optimized；The sequence of fnr1 gene is such as shown in SEQIDNO.5, the aminoacid sequence of fnr1 is such as shown in SEQIDNO.6, the sequence of fnr2 gene is such as shown in SEQIDNO.7, the aminoacid sequence of fnr2 is such as shown in SEQIDNO.8, the sequence of pKSC-fnr1 is such as shown in SEQIDNO.9, and the sequence of pKSC-fnr2 is such as shown in SEQIDNO.10；
3, coli strain is replaced with fnr1 and the fnr2 gene in pKSC-fnr1 and pKSC-fnr2 respectivelyE.coliWild type fnr gene in DH5 α, obtains the coli strain that alcohol resistance improvesE.coliDH5 α fnr::fnr1 andE.coliDH5αfnr::fnr2。
Below in conjunction with embodiment, the invention will be further described, but should not be construed as limitation of the present invention.If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1, a kind of method improving escherichia coli alcohol resistance, comprise the following steps:
1. by coli strainE.coliWild type fnr gene clone in DH5 α is to pKSC plasmid, obtain recombinant vector pKSC-fnr, the sequence of wild type fnr gene is such as shown in SEQIDNO.1, the aminoacid sequence of wild type fnr is such as shown in SEQIDNO.2, the sequence of pKSC plasmid is such as shown in SEQIDNO.3, and the sequence of recombinant vector pKSC-fnr is such as shown in SEQIDNO.4；
2. base is replaced: the fnr gene on recombinant vector pKSC-fnr obtain step (1) carries out base replacement: G164A, C182T, obtains the recombinant vector pKSC-fnr1 optimized；The sequence of fnr1 gene is such as shown in SEQIDNO.5, and the aminoacid sequence of fnr1 is such as shown in SEQIDNO.6, and the sequence of pKSC-fnr1 is such as shown in SEQIDNO.9；
3. replace coli strain with the fnr1 gene in pKSC-fnr1E.coliWild type fnr gene in DH5 α, replacement method is as follows:
3.1 connect the fnr1 gene in pKSC-fnr1 and kalamycin resistance gene respectively on TA cloning vehicle PMD-18, and contain the box gene of fnr1 gene and kalamycin resistance gene with restructuring homology arm primer amplification；The gene order of restructuring homology arm primer fnr_RED_for is such as shown in SEQIDNO.11；The gene order such as SEQIDNO.12 of restructuring homology arm primer fnr_RED_rev；Box gene sequence is such as shown in SEQIDNO.13；
3.2 these box genes are by escherichia coli recombination system (λ-red) restructuring to coli strainE.coliOn the wild type fnr gene of DH5 α, obtain optimizing bacterial strainE.coliDH5 α fnr::fnr1；
4. compare coli strainE.coliThe optimization bacterial strain that DH5 α and step 3 obtainE.coliThe alcohol resistance of DH5 α fnr::fnr1, step is as follows:
4.1 by coli strainE.coliThe optimization bacterial strain that DH5 α and step 3 obtainE.coliDH5 α fnr::fnr1 is inoculated in 50mlLB culture medium respectively, cultivates 12h for 37 DEG C, as seed liquor；
4.2 take 2 kinds of each 1ml of culture fluid that step (4.1) obtains respectively, are inoculated in 50mlLB culture medium respectively, cultivate 10h for 37 DEG C；
4.3 take, every 2h, 2 kinds of culture fluid that step (4.2) obtains, and measure its absorbance at 600nm place on spectrophotometer；Its growing state is as shown in Figure 2.
Embodiment 2, a kind of method improving escherichia coli alcohol resistance, step is as follows:
1. by coli strainE.coliWild type fnr gene clone in DH5 α is to pKSC plasmid, obtain recombinant vector pKSC-fnr, the sequence of wild type fnr gene is such as shown in SEQIDNO.1, the aminoacid sequence of wild type fnr is such as shown in SEQIDNO.2, the sequence of pKSC plasmid is such as shown in SEQIDNO.3, and the sequence of recombinant vector pKSC-fnr is such as shown in SEQIDNO.4；
2. base is replaced: the fnr gene on recombinant vector pKSC-fnr obtain step (1) carries out base replacement: C409G, G665C, obtains the recombinant vector pKSC-fnr2 optimized；The sequence of fnr2 gene is such as shown in SEQIDNO.7, and the aminoacid sequence of fnr2 is such as shown in SEQIDNO.8, and the sequence of pKSC-fnr2 is such as shown in SEQIDNO.10；
3. replace coli strain with the fnr2 gene in pKSC-fnr2E.coliWild type fnr gene in DH5 α, replacement method is as follows:
3.1 connect the fnr2 gene in pKSC-fnr2 and kalamycin resistance gene respectively on TA cloning vehicle PMD-18, and contain the box gene of fnr2 gene and kalamycin resistance gene with restructuring homology arm primer amplification；The gene order of restructuring homology arm primer fnr_RED_for is such as shown in SEQIDNO.11；The gene order such as SEQIDNO.12 of restructuring homology arm primer fnr_RED_rev；Box gene sequence is such as shown in SEQIDNO.14；
3.2 these box genes are by escherichia coli recombination system (λ-red) restructuring to coli strainE.coliOn the wild type fnr gene of DH5 α, obtain optimizing bacterial strainE.coliDH5 α fnr::fnr2；
4. compare coli strainE.coliThe optimization bacterial strain that DH5 α and step 3 obtainE.coliThe alcohol resistance of DH5 α fnr::fnr2, step is as follows:
4.1 by coli strainE.coliThe optimization bacterial strain that DH5 α and step 3 obtainE.coliDH5 α fnr::fnr2 is inoculated in 50mlLB culture medium respectively, cultivates 12h for 37 DEG C, as seed liquor；
4.2 take 2 kinds of each 1ml of culture fluid that step (4.1) obtains respectively, are inoculated in 50mlLB culture medium respectively, cultivate 10h for 37 DEG C；
4.3 take, every 2h, 2 kinds of culture fluid that step (4.2) obtains, and measure its absorbance at 600nm place on spectrophotometer；Its growing state is as shown in Figure 2.
SEQUENCELISTING
<110>Zhejiang University
<120>a kind of method improving escherichia coli alcohol resistance
<160>14
<170>PatentInversion3.3
<210>1
<211>753
<212>DNA
<213>wild type fnr gene order
<400>1
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<213>aminoacid sequence of wild type fnr
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MetIleProGluLysArgIleIleArgArgIleGlnSerGlyGlyCys
151015
AlaIleHisCysGlnAspCysSerIleSerGlnLeuCysIleProPhe
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ThrLeuAsnGluHisGluLeuAspGlnLeuAspAsnIleIleGluArg
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LysLysProIleGlnLysGlyGlnThrLeuPheLysAlaGlyAspGlu
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LeuLysSerLeuTyrAlaIleArgSerGlyThrIleLysSerTyrThr
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GluThrIleSerArgLeuLeuGlyArgPheGlnLysSerGlyMetLeu
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GlnLeuAlaGlyHisThrArgAsnValAla
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tctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacg4320
aggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccagg4380
atcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggcttt4440
ttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcg4500
atgagtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaat4560
aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgtt4620
aatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatag4680
gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgtt4740
gttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcga4800
aaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg4860
gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagct4920
tgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggc4980
gctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctt5040
aatgcgccgctacagggcgcgtcccattcgccaatccggatatagttcctcctttcagca5100
aaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcgg5160
tggcagcagccaactcagcttcctttcgggctttgtttagcagcctaggtattaatcaag5220
acgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggc5280
cctttcgtc5289
<210>4
<211>6429
<212>DNA
<213>artificial sequence
<400>4
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgcc240
attcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat300
tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggt360
tttcccagtcacgacgttgtaaaacgacggccagtgccaagcttttacccttaacaactt420
aagggttttcaaatagatagacatatatttacatctaatatcggaattctctgctgttaa480
ggtttgcttagacttacttgctccctaaaaagatgttaaaattgacaaatatcaggatcc540
attacggcttgagcagacctatgatcccggaaaagcgaattatacggcgcattcagtctg600
gcggttgtgctatccattgccaggattgcagcatcagccagctttgcatcccgttcacac660
tcaacgaacatgagcttgatcagcttgataatatcattgagcggaagaagcctattcaga720
aaggccagacgctgtttaaggctggtgatgaacttaaatcgctttatgccatccgctccg780
gtacgattaaaagttataccatcactgagcaaggcgacgagcaaatcactggtttccatt840
tagcaggcgacctggtgggatttgacgccatcggcagcggccatcacccgagcttcgcgc900
aggcgctggaaacctcgatggtatgtgaaatcccgttcgaaacgctggacgatttgtccg960
gtaaaatgccgaatctgcgtcagcagatgatgcgtctgatgagcggtgaaatcaaaggcg1020
atcaggacatgatcctgctgttgtcgaagaaaaatgccgaggaacgtctggctgcattca1080
tctacaacctgtcccgtcgttttgcccaacgcggcttctcccctcgtgaattccgcctga1140
cgatgactcgtggcgatatcggtaactatctgggcctgacggtagaaaccatcagccgtc1200
tgctgggtcgcttccagaaaagcggcatgctggcagtcaaaggtaaatacatcaccatcg1260
aaaataacgatgcgctggcccagcttgctggtcatacgcgtaacgttgcctgatttttcc1320
gcataactcactaggtacccaggcatcaaataaaacgaaaggctcagtcgaaagactggg1380
cctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgg1440
gagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccat1500
aaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttc1560
tacaaactcttcctgtcgtcatatctacagaattcgtaatcatggtcatagctgtttcct1620
gtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgt1680
aaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgccc1740
gctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggg1800
agaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg1860
gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca1920
gaatcaggggataacgcaggaaagaacatggacgtgtcagcggctttccagaactcggac1980
ggtttatgcgctgcccacgccggcatattgccggactccttgtgctcaaggtcggagtct2040
ttttcacgggcatactttccctcacgcgcaatataatcggcatgaggagaggcactgcct2100
tttccgccggtttttacgctgagatgataggatgccatcgtgttttatcccgctgaaggg2160
cgcacgtttctgaacgaagtgaagaaagtctaagtgcgccctgataaataaaagagttat2220
cagggattgtagtgggatttgacctcctctgccatcatgagcgtaatcattccgttagca2280
ttcaggaggtaaacagcatgaataaaagcgaaaaaacaggaacaatgggcagcagaaaga2340
gtgcagtatattcgcggcttaaagtcgccgaatgagcaacagaaacttatgctgatactg2400
acggataaagcagataaaacagcacaggatatcaaaacgctgtccctgctgatgaaggct2460
gaacaggcagcagagaaagcgcaggaagccagagcgaaagtcatgaacctgatacaggca2520
gaaaagcgagccgaagccagagccgcccgtaaagcccgtgaccatgctctgtaccagtct2580
gccggattgcttatcctggcgggtctggttgacagtaagacgggtaagcctgttgatgat2640
accgctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaag2700
tggtcagactggaaaatcagagggcaggaactgctgaacagcaaaaagtcagatagcacc2760
acatagcagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattat2820
gggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcac2880
tgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcct2940
gatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctactta3000
agcctttagggttttaaggtctgttttgtagaggagcaaacagcgtttgcgacatccttt3060
tgtaatactgcggaactgactaaagtagtgagttatacacagggctgggatctattcttt3120
ttatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaa3180
aaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccag3240
caaaggtctagcagaatttacagatacccacaactcaaaggaaaaggactagtaattatc3300
attgactagcccatctcaattggtatagtgattaaaatcacctagaccaattgagatgta3360
tgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacgg3420
agcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattgaaaacc3480
ctacaaggaaagaacggacggtatcgttcacttataaccaatacgctcagatgatgaaca3540
tcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaa3600
ctgtggaaatcaggaatcctttggttaaaggctttgagattttccagtggacaaactatg3660
ccaagttctcaagcgaaaaattagaattagtttttagtgaagagatattgccttatcttt3720
tccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaat3780
actctatgaggatttatgagtggttattaaaagaactaacacaaaagaaaactcacaagg3840
caaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactacc3900
atgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacactt3960
acagcaatatgaaattggtggttgataagcgaggccgcccgactgatacgttgattttcc4020
aagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaa4080
tgaatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaa4140
gaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaaat4200
ttttgagtgacatgcaaagtaagcatgatctcaatggttcgttctcatggctcacgcaaa4260
aacaacgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatg4320
gctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcac4380
cgttagatatcaaagggaaaactgtccatatgcacagatgaaaacggtgtaaaaaagata4440
gatacatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatc4500
gacaatgtaacagatgaacagcatgtaacacctaatagaacaggtgaaaccagtaaaaca4560
aagcaactagaacatgaaattgaacacctgagacaacttgttacagctcaacagtcacac4620
atagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggag4680
ccagtgacgcctcccgtggggaaaaaatcatctcgaggaagatcctttgatcttttctac4740
ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataa4800
aactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaa4860
cgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaat4920
gggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccg4980
atgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatg5040
agatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcatttta5100
tccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattcc5160
aggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcc5220
tgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttc5280
gtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatg5340
acgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccat5400
tctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacg5460
aggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccagg5520
atcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggcttt5580
ttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcg5640
atgagtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaat5700
aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgtt5760
aatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatag5820
gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgtt5880
gttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcga5940
aaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg6000
gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagct6060
tgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggc6120
gctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctt6180
aatgcgccgctacagggcgcgtcccattcgccaatccggatatagttcctcctttcagca6240
aaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcgg6300
tggcagcagccaactcagcttcctttcgggctttgtttagcagcctaggtattaatcaag6360
acgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggc6420
cctttcgtc6429
<210>5
<211>753
<212>DNA
<213>artificial sequence
<400>5
atgatcccggaaaagcgaattatacggcgcattcagtctggcggttgtgctatccattgc60
caggattgcagcatcagccagctttgcatcccgttcacactcaacgaacatgagcttgat120
cagcttgataatatcattgagcggaagaagcctattcagaaagaccagacgctgtttaag180
gttggtgatgaacttaaatcgctttatgccatccgctccggtacgattaaaagttatacc240
atcactgagcaaggcgacgagcaaatcactggtttccatttagcaggcgacctggtggga300
tttgacgccatcggcagcggccatcacccgagcttcgcgcaggcgctggaaacctcgatg360
gtatgtgaaatcccgttcgaaacgctggacgatttgtccggtaaaatgccgaatctgcgt420
cagcagatgatgcgtctgatgagcggtgaaatcaaaggcgatcaggacatgatcctgctg480
ttgtcgaagaaaaatgccgaggaacgtctggctgcattcatctacaacctgtcccgtcgt540
tttgcccaacgcggcttctcccctcgtgaattccgcctgacgatgactcgtggcgatatc600
ggtaactatctgggcctgacggtagaaaccatcagccgtctgctgggtcgcttccagaaa660
agcggcatgctggcagtcaaaggtaaatacatcaccatcgaaaataacgatgcgctggcc720
cagcttgctggtcatacgcgtaacgttgcctga753
<210>6
<211>250
<212>PRT
<213>artificial sequence
<400>6
MetIleProGluLysArgIleIleArgArgIleGlnSerGlyGlyCys
151015
AlaIleHisCysGlnAspCysSerIleSerGlnLeuCysIleProPhe
202530
ThrLeuAsnGluHisGluLeuAspGlnLeuAspAsnIleIleGluArg
354045
LysLysProIleGlnLysAspGlnThrLeuPheLysValGlyAspGlu
505560
LeuLysSerLeuTyrAlaIleArgSerGlyThrIleLysSerTyrThr
65707580
IleThrGluGlnGlyAspGluGlnIleThrGlyPheHisLeuAlaGly
859095
AspLeuValGlyPheAspAlaIleGlySerGlyHisHisProSerPhe
100105110
AlaGlnAlaLeuGluThrSerMetValCysGluIleProPheGluThr
115120125
LeuAspAspLeuSerGlyLysMetProAsnLeuArgGlnGlnMetMet
130135140
ArgLeuMetSerGlyGluIleLysGlyAspGlnAspMetIleLeuLeu
145150155160
LeuSerLysLysAsnAlaGluGluArgLeuAlaAlaPheIleTyrAsn
165170175
LeuSerArgArgPheAlaGlnArgGlyPheSerProArgGluPheArg
180185190
LeuThrMetThrArgGlyAspIleGlyAsnTyrLeuGlyLeuThrVal
195200205
GluThrIleSerArgLeuLeuGlyArgPheGlnLysSerGlyMetLeu
210215220
AlaValLysGlyLysTyrIleThrIleGluAsnAsnAspAlaLeuAla
225230235240
GlnLeuAlaGlyHisThrArgAsnValAla
245250
<210>7
<211>753
<212>DNA
<213>artificial sequence
<400>7
atgatcccggaaaagcgaattatacggcgcattcagtctggcggttgtgctatccattgc60
caggattgcagcatcagccagctttgcatcccgttcacactcaacgaacatgagcttgat120
cagcttgataatatcattgagcggaagaagcctattcagaaaggccagacgctgtttaag180
gctggtgatgaacttaaatcgctttatgccatccgctccggtacgattaaaagttatacc240
atcactgagcaaggcgacgagcaaatcactggtttccatttagcaggcgacctggtggga300
tttgacgccatcggcagcggccatcacccgagcttcgcgcaggcgctggaaacctcgatg360
gtatgtgaaatcccgttcgaaacgctggacgatttgtccggtaaaatggcgaatctgcgt420
cagcagatgatgcgtctgatgagcggtgaaatcaaaggcgatcaggacatgatcctgctg480
ttgtcgaagaaaaatgccgaggaacgtctggctgcattcatctacaacctgtcccgtcgt540
tttgcccaacgcggcttctcccctcgtgaattccgcctgacgatgactcgtggcgatatc600
ggtaactatctgggcctgacggtagaaaccatcagccgtctgctgggtcgcttccagaaa660
agcgccatgctggcagtcaaaggtaaatacatcaccatcgaaaataacgatgcgctggcc720
cagcttgctggtcatacgcgtaacgttgcctga753
<210>8
<211>250
<212>PRT
<213>artificial sequence
<400>8
MetIleProGluLysArgIleIleArgArgIleGlnSerGlyGlyCys
151015
AlaIleHisCysGlnAspCysSerIleSerGlnLeuCysIleProPhe
202530
ThrLeuAsnGluHisGluLeuAspGlnLeuAspAsnIleIleGluArg
354045
LysLysProIleGlnLysGlyGlnThrLeuPheLysAlaGlyAspGlu
505560
LeuLysSerLeuTyrAlaIleArgSerGlyThrIleLysSerTyrThr
65707580
IleThrGluGlnGlyAspGluGlnIleThrGlyPheHisLeuAlaGly
859095
AspLeuValGlyPheAspAlaIleGlySerGlyHisHisProSerPhe
100105110
AlaGlnAlaLeuGluThrSerMetValCysGluIleProPheGluThr
115120125
LeuAspAspLeuSerGlyLysMetAlaAsnLeuArgGlnGlnMetMet
130135140
ArgLeuMetSerGlyGluIleLysGlyAspGlnAspMetIleLeuLeu
145150155160
LeuSerLysLysAsnAlaGluGluArgLeuAlaAlaPheIleTyrAsn
165170175
LeuSerArgArgPheAlaGlnArgGlyPheSerProArgGluPheArg
180185190
LeuThrMetThrArgGlyAspIleGlyAsnTyrLeuGlyLeuThrVal
195200205
GluThrIleSerArgLeuLeuGlyArgPheGlnLysSerAlaMetLeu
210215220
AlaValLysGlyLysTyrIleThrIleGluAsnAsnAspAlaLeuAla
225230235240
GlnLeuAlaGlyHisThrArgAsnValAla
245250
<210>9
<211>6429
<212>DNA
<213>artificial sequence
<400>9
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgcc240
attcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat300
tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggt360
tttcccagtcacgacgttgtaaaacgacggccagtgccaagcttttacccttaacaactt420
aagggttttcaaatagatagacatatatttacatctaatatcggaattctctgctgttaa480
ggtttgcttagacttacttgctccctaaaaagatgttaaaattgacaaatatcaggatcc540
attacggcttgagcagacctatgatcccggaaaagcgaattatacggcgcattcagtctg600
gcggttgtgctatccattgccaggattgcagcatcagccagctttgcatcccgttcacac660
tcaacgaacatgagcttgatcagcttgataatatcattgagcggaagaagcctattcaga720
aagaccagacgctgtttaaggttggtgatgaacttaaatcgctttatgccatccgctccg780
gtacgattaaaagttataccatcactgagcaaggcgacgagcaaatcactggtttccatt840
tagcaggcgacctggtgggatttgacgccatcggcagcggccatcacccgagcttcgcgc900
aggcgctggaaacctcgatggtatgtgaaatcccgttcgaaacgctggacgatttgtccg960
gtaaaatgccgaatctgcgtcagcagatgatgcgtctgatgagcggtgaaatcaaaggcg1020
atcaggacatgatcctgctgttgtcgaagaaaaatgccgaggaacgtctggctgcattca1080
tctacaacctgtcccgtcgttttgcccaacgcggcttctcccctcgtgaattccgcctga1140
cgatgactcgtggcgatatcggtaactatctgggcctgacggtagaaaccatcagccgtc1200
tgctgggtcgcttccagaaaagcggcatgctggcagtcaaaggtaaatacatcaccatcg1260
aaaataacgatgcgctggcccagcttgctggtcatacgcgtaacgttgcctgatttttcc1320
gcataactcactaggtacccaggcatcaaataaaacgaaaggctcagtcgaaagactggg1380
cctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgg1440
gagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccat1500
aaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttc1560
tacaaactcttcctgtcgtcatatctacagaattcgtaatcatggtcatagctgtttcct1620
gtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgt1680
aaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgccc1740
gctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggg1800
agaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg1860
gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca1920
gaatcaggggataacgcaggaaagaacatggacgtgtcagcggctttccagaactcggac1980
ggtttatgcgctgcccacgccggcatattgccggactccttgtgctcaaggtcggagtct2040
ttttcacgggcatactttccctcacgcgcaatataatcggcatgaggagaggcactgcct2100
tttccgccggtttttacgctgagatgataggatgccatcgtgttttatcccgctgaaggg2160
cgcacgtttctgaacgaagtgaagaaagtctaagtgcgccctgataaataaaagagttat2220
cagggattgtagtgggatttgacctcctctgccatcatgagcgtaatcattccgttagca2280
ttcaggaggtaaacagcatgaataaaagcgaaaaaacaggaacaatgggcagcagaaaga2340
gtgcagtatattcgcggcttaaagtcgccgaatgagcaacagaaacttatgctgatactg2400
acggataaagcagataaaacagcacaggatatcaaaacgctgtccctgctgatgaaggct2460
gaacaggcagcagagaaagcgcaggaagccagagcgaaagtcatgaacctgatacaggca2520
gaaaagcgagccgaagccagagccgcccgtaaagcccgtgaccatgctctgtaccagtct2580
gccggattgcttatcctggcgggtctggttgacagtaagacgggtaagcctgttgatgat2640
accgctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaag2700
tggtcagactggaaaatcagagggcaggaactgctgaacagcaaaaagtcagatagcacc2760
acatagcagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattat2820
gggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcac2880
tgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcct2940
gatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctactta3000
agcctttagggttttaaggtctgttttgtagaggagcaaacagcgtttgcgacatccttt3060
tgtaatactgcggaactgactaaagtagtgagttatacacagggctgggatctattcttt3120
ttatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaa3180
aaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccag3240
caaaggtctagcagaatttacagatacccacaactcaaaggaaaaggactagtaattatc3300
attgactagcccatctcaattggtatagtgattaaaatcacctagaccaattgagatgta3360
tgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacgg3420
agcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattgaaaacc3480
ctacaaggaaagaacggacggtatcgttcacttataaccaatacgctcagatgatgaaca3540
tcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaa3600
ctgtggaaatcaggaatcctttggttaaaggctttgagattttccagtggacaaactatg3660
ccaagttctcaagcgaaaaattagaattagtttttagtgaagagatattgccttatcttt3720
tccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaat3780
actctatgaggatttatgagtggttattaaaagaactaacacaaaagaaaactcacaagg3840
caaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactacc3900
atgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacactt3960
acagcaatatgaaattggtggttgataagcgaggccgcccgactgatacgttgattttcc4020
aagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaa4080
tgaatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaa4140
gaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaaat4200
ttttgagtgacatgcaaagtaagcatgatctcaatggttcgttctcatggctcacgcaaa4260
aacaacgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatg4320
gctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcac4380
cgttagatatcaaagggaaaactgtccatatgcacagatgaaaacggtgtaaaaaagata4440
gatacatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatc4500
gacaatgtaacagatgaacagcatgtaacacctaatagaacaggtgaaaccagtaaaaca4560
aagcaactagaacatgaaattgaacacctgagacaacttgttacagctcaacagtcacac4620
atagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggag4680
ccagtgacgcctcccgtggggaaaaaatcatctcgaggaagatcctttgatcttttctac4740
ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataa4800
aactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaa4860
cgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaat4920
gggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccg4980
atgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatg5040
agatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcatttta5100
tccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattcc5160
aggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcc5220
tgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttc5280
gtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatg5340
acgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccat5400
tctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacg5460
aggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccagg5520
atcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggcttt5580
ttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcg5640
atgagtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaat5700
aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgtt5760
aatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatag5820
gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgtt5880
gttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcga5940
aaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg6000
gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagct6060
tgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggc6120
gctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctt6180
aatgcgccgctacagggcgcgtcccattcgccaatccggatatagttcctcctttcagca6240
aaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcgg6300
tggcagcagccaactcagcttcctttcgggctttgtttagcagcctaggtattaatcaag6360
acgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggc6420
cctttcgtc6429
<210>10
<211>6429
<212>DNA
<213>artificial sequence
<400>10
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca60
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg120
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc180
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgcc240
attcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctat300
tacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggt360
tttcccagtcacgacgttgtaaaacgacggccagtgccaagcttttacccttaacaactt420
aagggttttcaaatagatagacatatatttacatctaatatcggaattctctgctgttaa480
ggtttgcttagacttacttgctccctaaaaagatgttaaaattgacaaatatcaggatcc540
attacggcttgagcagacctatgatcccggaaaagcgaattatacggcgcattcagtctg600
gcggttgtgctatccattgccaggattgcagcatcagccagctttgcatcccgttcacac660
tcaacgaacatgagcttgatcagcttgataatatcattgagcggaagaagcctattcaga720
aaggccagacgctgtttaaggctggtgatgaacttaaatcgctttatgccatccgctccg780
gtacgattaaaagttataccatcactgagcaaggcgacgagcaaatcactggtttccatt840
tagcaggcgacctggtgggatttgacgccatcggcagcggccatcacccgagcttcgcgc900
aggcgctggaaacctcgatggtatgtgaaatcccgttcgaaacgctggacgatttgtccg960
gtaaaatggcgaatctgcgtcagcagatgatgcgtctgatgagcggtgaaatcaaaggcg1020
atcaggacatgatcctgctgttgtcgaagaaaaatgccgaggaacgtctggctgcattca1080
tctacaacctgtcccgtcgttttgcccaacgcggcttctcccctcgtgaattccgcctga1140
cgatgactcgtggcgatatcggtaactatctgggcctgacggtagaaaccatcagccgtc1200
tgctgggtcgcttccagaaaagcgccatgctggcagtcaaaggtaaatacatcaccatcg1260
aaaataacgatgcgctggcccagcttgctggtcatacgcgtaacgttgcctgatttttcc1320
gcataactcactaggtacccaggcatcaaataaaacgaaaggctcagtcgaaagactggg1380
cctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgg1440
gagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccat1500
aaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttc1560
tacaaactcttcctgtcgtcatatctacagaattcgtaatcatggtcatagctgtttcct1620
gtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgt1680
aaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgccc1740
gctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggg1800
agaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcg1860
gtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca1920
gaatcaggggataacgcaggaaagaacatggacgtgtcagcggctttccagaactcggac1980
ggtttatgcgctgcccacgccggcatattgccggactccttgtgctcaaggtcggagtct2040
ttttcacgggcatactttccctcacgcgcaatataatcggcatgaggagaggcactgcct2100
tttccgccggtttttacgctgagatgataggatgccatcgtgttttatcccgctgaaggg2160
cgcacgtttctgaacgaagtgaagaaagtctaagtgcgccctgataaataaaagagttat2220
cagggattgtagtgggatttgacctcctctgccatcatgagcgtaatcattccgttagca2280
ttcaggaggtaaacagcatgaataaaagcgaaaaaacaggaacaatgggcagcagaaaga2340
gtgcagtatattcgcggcttaaagtcgccgaatgagcaacagaaacttatgctgatactg2400
acggataaagcagataaaacagcacaggatatcaaaacgctgtccctgctgatgaaggct2460
gaacaggcagcagagaaagcgcaggaagccagagcgaaagtcatgaacctgatacaggca2520
gaaaagcgagccgaagccagagccgcccgtaaagcccgtgaccatgctctgtaccagtct2580
gccggattgcttatcctggcgggtctggttgacagtaagacgggtaagcctgttgatgat2640
accgctgccttactgggtgcattagccagtctgaatgacctgtcacgggataatccgaag2700
tggtcagactggaaaatcagagggcaggaactgctgaacagcaaaaagtcagatagcacc2760
acatagcagacccgccataaaacgccctgagaagcccgtgacgggcttttcttgtattat2820
gggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcac2880
tgccagagccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcct2940
gatggtcggagacaaaaggaatattcagcgatttgcccgagcttgcgagggtgctactta3000
agcctttagggttttaaggtctgttttgtagaggagcaaacagcgtttgcgacatccttt3060
tgtaatactgcggaactgactaaagtagtgagttatacacagggctgggatctattcttt3120
ttatctttttttattctttctttattctataaattataaccacttgaatataaacaaaaa3180
aaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccag3240
caaaggtctagcagaatttacagatacccacaactcaaaggaaaaggactagtaattatc3300
attgactagcccatctcaattggtatagtgattaaaatcacctagaccaattgagatgta3360
tgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacgg3420
agcatgaaaccaagctaattttatgctgtgtggcactactcaaccccacgattgaaaacc3480
ctacaaggaaagaacggacggtatcgttcacttataaccaatacgctcagatgatgaaca3540
tcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaa3600
ctgtggaaatcaggaatcctttggttaaaggctttgagattttccagtggacaaactatg3660
ccaagttctcaagcgaaaaattagaattagtttttagtgaagagatattgccttatcttt3720
tccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaat3780
actctatgaggatttatgagtggttattaaaagaactaacacaaaagaaaactcacaagg3840
caaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactacc3900
atgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacactt3960
acagcaatatgaaattggtggttgataagcgaggccgcccgactgatacgttgattttcc4020
aagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaa4080
tgaatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaa4140
gaaaaacactacacgatgctttaactgcaaaaattcagctcaccagttttgaggcaaaat4200
ttttgagtgacatgcaaagtaagcatgatctcaatggttcgttctcatggctcacgcaaa4260
aacaacgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatg4320
gctcttgtatctatcagtgaagcatcaagactaacaaacaaaagtagaacaactgttcac4380
cgttagatatcaaagggaaaactgtccatatgcacagatgaaaacggtgtaaaaaagata4440
gatacatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatc4500
gacaatgtaacagatgaacagcatgtaacacctaatagaacaggtgaaaccagtaaaaca4560
aagcaactagaacatgaaattgaacacctgagacaacttgttacagctcaacagtcacac4620
atagacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggag4680
ccagtgacgcctcccgtggggaaaaaatcatctcgaggaagatcctttgatcttttctac4740
ggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataa4800
aactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaa4860
cgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaat4920
gggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccg4980
atgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatg5040
agatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcatttta5100
tccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattcc5160
aggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcc5220
tgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttc5280
gtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatg5340
acgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccat5400
tctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacg5460
aggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccagg5520
atcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggcttt5580
ttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcg5640
atgagtttttctaagaattaattcatgagcggatacatatttgaatgtatttagaaaaat5700
aaacaaataggggttccgcgcacatttccccgaaaagtgccacctgaaattgtaaacgtt5760
aatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatag5820
gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgtt5880
gttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcga5940
aaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttg6000
gggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagct6060
tgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggc6120
gctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctt6180
aatgcgccgctacagggcgcgtcccattcgccaatccggatatagttcctcctttcagca6240
aaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcgg6300
tggcagcagccaactcagcttcctttcgggctttgtttagcagcctaggtattaatcaag6360
acgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggc6420
cctttcgtc6429
<210>11
<211>81
<212>DNA
<213>artificial sequence
<400>11
cctaaaaagatgttaaaattgacaaatatcaattacggcttgagcagacctatgatcccg60
gaaaagcgaattatacggcgc81
<210>12
<211>81
<212>DNA
<213>artificial sequence
<400>12
agaaaaatttaatgatatgacagaaggatagtgagttatgcggaaaaatcatgagcgatt60
gtgtaggctggagctgcttcg81
<210>13
<211>1808
<212>DNA
<213>artificial sequence
<400>13
atgatcccggaaaagcgaattatacggcgcattcagtctggcggttgtgctatccattgc60
caggattgcagcatcagccagctttgcatcccgttcacactcaacgaacatgagcttgat120
cagcttgataatatcattgagcggaagaagcctattcagaaagaccagacgctgtttaag180
gttggtgatgaacttaaatcgctttatgccatccgctccggtacgattaaaagttatacc240
atcactgagcaaggcgacgagcaaatcactggtttccatttagcaggcgacctggtggga300
tttgacgccatcggcagcggccatcacccgagcttcgcgcaggcgctggaaacctcgatg360
gtatgtgaaatcccgttcgaaacgctggacgatttgtccggtaaaatgccgaatctgcgt420
cagcagatgatgcgtctgatgagcggtgaaatcaaaggcgatcaggacatgatcctgctg480
ttgtcgaagaaaaatgccgaggaacgtctggctgcattcatctacaacctgtcccgtcgt540
tttgcccaacgcggcttctcccctcgtgaattccgcctgacgatgactcgtggcgatatc600
ggtaactatctgggcctgacggtagaaaccatcagccgtctgctgggtcgcttccagaaa660
agcggcatgctggcagtcaaaggtaaatacatcaccatcgaaaataacgatgcgctggcc720
cagcttgctggtcatacgcgtaacgttgcctgaaagcttcatgggaattagccatggtcc780
atatgaatatcctccttagttcctattccgaagttcctattctctagaaagtataggaac840
ttcggcgcgcctacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccc900
tgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgt960
aagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagtt1020
gtcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccac1080
cgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctca1140
atgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaa1200
aaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctca1260
tccggaattacgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaccc1320
ttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaatacca1380
cgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaa1440
cctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctg1500
ggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgt1560
tttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattca1620
ggttcatcatgccgtttgtgatggcttccatgtcggcagatgcttaatgaatacaacagt1680
actgcgatgagtggcagggcggggcgtaaggcgcgccatttaaatgaagttcctattccg1740
aagttcctattctctagaaagtataggaacttcgaagcagctccagcctacacaatcgct1800
caaagctt1808
<210>14
<211>1808
<212>DNA
<213>artificial sequence
<400>14
atgatcccggaaaagcgaattatacggcgcattcagtctggcggttgtgctatccattgc60
caggattgcagcatcagccagctttgcatcccgttcacactcaacgaacatgagcttgat120
cagcttgataatatcattgagcggaagaagcctattcagaaaggccagacgctgtttaag180
gctggtgatgaacttaaatcgctttatgccatccgctccggtacgattaaaagttatacc240
atcactgagcaaggcgacgagcaaatcactggtttccatttagcaggcgacctggtggga300
tttgacgccatcggcagcggccatcacccgagcttcgcgcaggcgctggaaacctcgatg360
gtatgtgaaatcccgttcgaaacgctggacgatttgtccggtaaaatggcgaatctgcgt420
cagcagatgatgcgtctgatgagcggtgaaatcaaaggcgatcaggacatgatcctgctg480
ttgtcgaagaaaaatgccgaggaacgtctggctgcattcatctacaacctgtcccgtcgt540
tttgcccaacgcggcttctcccctcgtgaattccgcctgacgatgactcgtggcgatatc600
ggtaactatctgggcctgacggtagaaaccatcagccgtctgctgggtcgcttccagaaa660
agcgccatgctggcagtcaaaggtaaatacatcaccatcgaaaataacgatgcgctggcc720
cagcttgctggtcatacgcgtaacgttgcctgaaagcttcatgggaattagccatggtcc780
atatgaatatcctccttagttcctattccgaagttcctattctctagaaagtataggaac840
ttcggcgcgcctacctgtgacggaagatcacttcgcagaataaataaatcctggtgtccc900
tgttgataccgggaagccctgggccaacttttggcgaaaatgagacgttgatcggcacgt960
aagaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagtt1020
gtcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccac1080
cgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctca1140
atgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaa1200
aaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctca1260
tccggaattacgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaccc1320
ttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaatacca1380
cgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaa1440
cctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctg1500
ggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgt1560
tttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattca1620
ggttcatcatgccgtttgtgatggcttccatgtcggcagatgcttaatgaatacaacagt1680
actgcgatgagtggcagggcggggcgtaaggcgcgccatttaaatgaagttcctattccg1740
aagttcctattctctagaaagtataggaacttcgaagcagctccagcctacacaatcgct1800
caaagctt1808
Claims (1)
1. the method improving escherichia coli alcohol resistance, it is characterised in that comprise the following steps:
(1) by coli strain (E.coli) wild type fnr gene clone in DH5 α is to pKSC plasmid, obtain recombinant vector pKSC-fnr, the sequence of wild type fnr gene is such as shown in SEQIDNO.1, the aminoacid sequence of wild type fnr is such as shown in SEQIDNO.2, the sequence of pKSC plasmid is such as shown in SEQIDNO.3, and the sequence of recombinant vector pKSC-fnr is such as shown in SEQIDNO.4；
(2) base is replaced: the fnr gene on recombinant vector pKSC-fnr step 1 obtained carries out base replacement: G164A, C182T, obtains the recombinant vector pKSC-fnr1 optimized；Base is replaced: C409G, G665C, obtains the recombinant vector pKSC-fnr2 optimized；The sequence of fnr1 gene is such as shown in SEQIDNO.5, the aminoacid sequence of fnr1 is such as shown in SEQIDNO.6, the sequence of fnr2 gene is such as shown in SEQIDNO.7, the aminoacid sequence of fnr2 is such as shown in SEQIDNO.8, the sequence of pKSC-fnr1 is such as shown in SEQIDNO.9, and the sequence of pKSC-fnr2 is such as shown in SEQIDNO.10；
(3) replace the wild type fnr gene in coli strain DH5 α with fnr1 and the fnr2 gene in pKSC-fnr1 and pKSC-fnr2 respectively, obtain coli strain DH5 α fnr::fnr1 and DH5 α fnr::fnr2 that alcohol resistance improves.
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| US20100255036A1 (en) * | 2006-07-19 | 2010-10-07 | North Carolina State University | Attenuated fnr deficient enterobacteria |
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Non-Patent Citations (4)
| Title |
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| FNR转录因子定向进化提高大肠杆菌对乙醇和丁醇的耐受性;朱森康;《中国优秀硕士学位论文全文数据库（基础科学辑）》;20120615(第6期);全文 * |
| Metabolic Engineering of Escherichia coli for production of Mixed-Acid Fermentation End Products;Andreas H.F.等;《Front Bioeng Biotechnol》;20140523;第2卷(第16期);全文 * |
| Reactions of nitric oxide and oxygen with the regulator of fumarate and nitrate reduction, a global transcriptional regulator, during anaerobic growth of Escherichia coli.;Crack JC 等;《Methods Enzymol》;20081231;全文 * |
| Transcription Activation at Escherichia coli FNR-Dependent Promoters by the Gonococcal FNR protein: Effects of a Novel S18F substitution and Comparisons with the Corresponding Substitution in E. coli FNR;Tim Overton 等;《J Bacteriol》;20030831;第185卷(第16期);全文 * |
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