CN103898098A - Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof - Google Patents
Escherichia coli-clostridium shuttle expression vector, and construction and expression thereof Download PDFInfo
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Abstract
The invention discloses an escherichia coli-clostridium shuttle expression vector, and construction and expression thereof. The escherichia coli-clostridium shuttle expression vector is constructed by inserting escherichia coli lacZ gene (EclacZS) coding sequence between BamHI and SacI restriction enzyme cutting sites of pYN7443 plasmid, wherein the escherichia coli lacZ gene (EclacZS) coding sequence is obtained via clostridium preferred codon chemical synthesis. It is confirmed by escherichia coli lacZ gene activity determination on clostridium transformed from the escherichia coli-clostridium shuttle expression vector that the lacZ gene, which comes from escherichia coli and is obtained via clostridium preferred codon chemical synthesis, is a report gene suitable for expresion in clostridium. Construction of high butanol yield engineered strains via determination of optimal promoter-terminator combination possesses important theoretical significance, wherein determination of the optimal promoter-terminator combination is realized by comparing different promoters and terminators.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of intestinal bacteria-clostridium shuttle expression carrier and structure and expression, be specifically related to a kind ofly according to intestinal bacteria-clostridium shuttle expression carrier and the structure thereof of the chemosynthesis of clostridium codon preference, and this expression vector can be successfully seen to the application that intestinal bacteria lacZ gene is expressed in clostridium being transformed in clostridium.
Background technology
Although obtained sizable achievement at the metabolic engineering of clostridium in recent years, but not good the further developing of limiting acetone butanol fermentation of the research of several necessary genetic tools, one of them most important genetic tool is exactly the reporter gene of controlling exogenous gene expression.A good reporter gene can help different homologies or the allogeneic promoter in the detailed research solvent clostridium of investigator, thereby can further understand the adjusting function of these promotors in clostridium.Only have intensity and the adjusting function of understanding better promotor just can produce more effectively clostridium expression vector, such expression vector finally can be used as improving the solvent production of acetone butanol fermentation.Understanding under the intensity of different promoters and the prerequisite of regulation and control, can develop more complicated engineering strain and improve the output of solvent in conjunction with Antisense RNA Technique and gene Knockout.
Owing to not having at present effective clostridium exogenous gene expression reporting system, so just there is no the research of detailed clostridium promotor at present.And that bacillus coli gene is expressed in clostridium is very undesirable, thereby traditional intestinal bacteria wild-type reporter gene just can not well use in clostridium.In other many bacterial strains, use very good reporter gene--green fluorescent protein for another, produce the chromophoric group of fluorescence because it needs enough oxygen, so green fluorescent protein is also not suitable for clostridium as reporter gene.Be suitable as very much the clostridium perfringens E.C. 2.3.1.28 of clostridium reporter gene because clostridium contains many nonspecific acetyl-CoAs of high level that may disturb E.C. 2.3.1.28 to express, thereby application prospect neither be very desirable.
Summary of the invention
One of technical problem to be solved by this invention is to provide a kind of intestinal bacteria lacZ gene, and its nucleotide sequence is as shown in SEQ ID No.2.By its called after EclacZS gene, it is by clostridium preference codon biological process transformation synthetic.
Two of technical problem to be solved by this invention is to provide a kind of promotor of clostridium OABC gene, and its nucleotide sequence is as shown in SEQ ID No.2.This promotor is with R27320(5 '-TCT, AGA, AAA, GGA, AAA, TAT, GAT, AAA, AAA, TTT, CA-3 ') and R27321(5 '-GGA, TCC, CAT, TAA, TAT, CGA, AAA, TAG, CTT, AAA, CCC, AAC-3 ') be primer, take the genomic dna (GenBank:AE001437.1) of clostridium as template, with the high-fidelity high temperature-resisting DNA polymerase Pyrobest of Japanese TAKARA company through pcr amplification and.
Three of technical problem to be solved by this invention is to provide a kind of pYN7443 plasmid of the promotor that comprises above-mentioned clostridium OABC gene, it is to form by original ptb gene promoter being replaced with to above-mentioned clostridium OABC gene promoter structure on the preparation method's of existing carrier pSOS94 basis, wherein pSOS94 preparation method is referring to U-B.Tummal et al., 1999, Applied Ana Environmental Microbiology, 9,3793 – 3799).
Four of technical problem to be solved by this invention is to provide a kind of intestinal bacteria-clostridium shuttle expression carrier (pYN7443-EclacZS), and it is that the sequence construct that inserts EclacZS genes encoding as above between the BamHI of above-mentioned pYN7443 plasmid and SacI restriction enzyme site forms.
Five of technical problem to be solved by this invention is to provide the construction process of a kind of described intestinal bacteria-clostridium shuttle expression carrier (pYN7443-EclacZS), specifically comprises the following steps:
1) according to clostridium preference password synthesising biological method [Ai-Sheng Xiong, Quan-hong Yao, Ri-He Peng, Xian Li, Huiqin Fan, Zong-ming Cheng and Yi Li, Nucleic Acid Research, 2004,32 (12), e98:1～10] design primer, primer is 5 ' end to 3 ' end, and then synthetic intestinal bacteria lacZ gene;
2) the pYN7443 plasmid of the promotor that structure comprises clostridium OABC gene;
3) utilize BamHI and SacI to carry out after double digestion, be that EclacZS sequence is connected with pYN7443 plasmid by T4DNA ligase enzyme by intestinal bacteria lacZ gene, and then obtain intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS.
This intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS is transformed in clostridium and measures the activity of intestinal bacteria lacZ gene, concrete steps are as follows:
1) transform clostridium with reference to the electric-shocking method (C Tardif et al., 2001, Journal ofIndustrial Microbiology & Biotechnology, 27,271-274) of Tardif etc.
2) carry out the determination of activity of intestinal bacteria lacZ gene with reference to the method (U-B.Tummal et al., 1999, Applied Ana Environmental Microbiology, 9,3793-3799) of U B.Tummal etc.
For the considerably less situation of adaptable reporter gene in current clostridium, the present invention has synthesized one according to clostridium codon preference and has derived from colibacillary LacZ gene, and having built a kind of novel intestinal bacteria-clostridium shuttle expression carrier, the determination of activity of carrying out intestinal bacteria lacZ gene by the clostridium that this carrier is transformed has proved that of the present invention is equally also a reporter gene that is suitable for expressing in clostridium according to the synthetic colibacillary LacZ gene that derives from of clostridium codon preference.This is for determining that by relatively different promoters and terminator the engineering strain that optimum promotor terminator combines to build high yield butanols has important theory significance.
Accompanying drawing explanation
Fig. 1 is the building mode of intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS of the present invention.
Fig. 2 is coated on the growing state on the flat board that contains erythromycin and X-gal after intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS of the present invention transforms clostridium.
Fig. 3 is the beta-galactosidase enzymes enzymic activity of different time sections.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.The embodiment of the present invention is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
If the reagent unexplained reference that the present invention is used, all purchased from Sigma-aldrich (Sigma-Aldrich) company.
The present invention relates to molecular biology experiment, as not dated especially, all with reference to from " molecular cloning "-book (J. Pehanorm Brooker, E.F. be Ritchie, T. Manny A Disi work not, 1994, Science Press).
Synthesizing of embodiment 1 intestinal bacteria lacZ gene
According to clostridium (GenBank:AE001437.1) preference codon synthesising biological method [Ai-Sheng Xiong, Quan-Hhong Yao, Ri-He Peng, Xian Li, Huiqin Fan, Zong-ming Cheng and Yi Li, Nucleic Acid Research, 2004,32 (12), e98:1～10] design following primer, wherein primer is 5 ' end to 3 ' end, and then optimizes synthetic intestinal bacteria lacZ gene.
1.EMPII-1:Tm=54,60mer
GGA,TCC,ATG,ACT,ATG,ATT,ACT,GAT,TCT,CTT,GCT,GTT,GTT,TTA,CAA,AGA,AGA,GAT,TGG,GAA
2.EMPII-2:Tm=54,60mer
GAT,GTG,CAG,CAA,GTC,TAT,TAA,GTT,GAG,TAA,CAC,CAG,GAT,TTT,CCC,AAT,CTC,TTC,TTT,GTA
3.EMPII-3:Tm=54,60mer
TAA,TAG,ACT,TGC,TGC,ACA,TCC,TCC,TTT,TGC,TTC,TTG,GAG,AAA,TTC,TGA,AGA,AGC,TAG,AAC
4.EMPII-4:Tm=54,60mer
TTC,ACC,ATT,AAG,TGA,TCT,AAG,TTG,TTG,AGA,TGG,TCT,ATC,AGT,TCT,AGC,TTC,TTC,AGA,ATT
5.EMPII-5:Tm=54,60mer
TTA,GAT,CAC,TTA,ATG,GTG,AAT,GGA,GAT,TTG,CAT,GGT,TTC,CAG,CAC,CAG,AAG,CAG,TTC,CTG
6.EMPII-6:Tm=54,60mer
ACA,GTA,TCA,GCT,TCA,GGA,AGA,TCA,CAT,TCA,AGC,CAA,GAT,TCA,GGA,ACT,GCT,TCT,GGT,GCT
7.EMPII-7:Tm=54,60mer
CTT,CCT,GAA,GCT,GAT,ACT,GTT,GTT,GTT,CCA,TCA,AAT,TGG,CAA,ATG,CAT,GGT,TAT,GAT,GCA
8.EMPII-8:Tm=54,60mer
GAT,TAA,CAG,TAA,TTG,GAT,AAG,TAA,CAT,TAG,TAT,AAA,TTG,GTG,CAT,CAT,AAC,CAT,GCA,TTT
9.EMPII-9:Tm=54,60mer
TTA,TCC,AAT,TAC,TGT,TAA,TCC,ACC,ATT,TGT,TCC,TAC,TGA,AAA,TCC,AAC,TGG,TTG,TTA,TTC
10.EMPII-10:Tm=54,60mer
ACC,TTC,TTG,TAG,CCA,AGA,TTC,ATC,AAC,ATT,AAA,AGT,AAG,AGA,ATA,ACA,ACC,AGT,TGG,ATT
11.EMPII-11:Tm=54,60mer
AAT,CTT,GGC,TAC,AAG,AAG,GTC,AAA,CTA,GAA,TTA,TTT,TTG,ATG,GTG,TTA,ATT,CTG,CAT,TTC
12.EMPII-12:Tm=54,60mer
TCT,TGA,CCA,TAA,CCA,ACC,CAT,CTA,CCA,TTA,CAC,CAA,AGA,TGA,AAT,GCA,GAA,TTA,ACA,CCA
13.EMPII-13:Tm=54,60mer
TGG,GTT,GGT,TAT,GGT,CAA,GAT,AGT,AGA,TTG,CCA,TCT,GAG,TTC,GAT,CTT,AGT,GCA,TTC,TTA
14.EMPII-14:Tm=54,60mer
ATC,TAA,GAA,CCA,TAA,CAG,CAA,GTC,TAT,TTT,CTC,CAG,CTC,TTA,AGA,ATG,CAC,TAA,GAT,CGA
15.EMPII-15:Tm=54,60mer
TGC,TGT,TAT,GGT,TCT,TAG,ATG,GAG,TGA,TGG,TAG,TTA,TCT,TGA,AGA,TCA,AGA,TAT,GTG,GAG
16.EMPII-16:Tm=54,60mer
CTT,ATG,AAG,CAA,AGA,AAC,ATC,TCT,GAA,AAT,ACC,ACT,CAT,TCT,CCA,CAT,ATC,TTG,ATC,TTC
17.EMPII-17:Tm=54,60mer
ATG,TTT,CTT,TGC,TTC,ATA,AGC,CTA,CTA,CAC,AAA,TTA,GTG,ATT,TCC,ATG,TTG,CTA,CTA,GAT
18.EMPII-18:Tm=54,60mer
ACT,TCA,GCT,TCC,AAA,ACA,GCT,CTA,CTG,AAA,TCA,TCA,TTA,AAT,CTA,GTA,GCA,ACA,TGG,AAA
19.EMPII-19:Tm=54,60mer
GCT,GTT,TTG,GAA,GCT,GAA,GTT,CAA,ATG,TGT,GGT,GAA,TTG,AGA,GAT,TAT,CTT,AGA,GTA,ACA
20.EMPII-20:Tm=54,60mer
TAC,CAC,TAG,CAA,CTT,GAG,TTT,CAC,CTT,GCC,ATA,AAG,AAA,CTG,TTA,CTC,TAA,GAT,AAT,CTC
21.EMPII-21:Tm=54,60mer
AAC,TCA,AGT,TGC,TAG,TGG,TAC,TGC,ACC,TTT,CGG,TGG,TGA,AAT,TAT,TGA,TGA,AAG,AGG,AGG
22.EMPII-22:Tm=54,60mer
ATT,TTC,AAC,ATT,AAG,TCT,AAG,TGT,AAC,TCT,ATC,TGC,ATA,TCC,TCC,TCT,TTC,ATC,AAT,AAT
23.EMPII-23:Tm=54,60mer
TTA,GAC,TTA,ATG,TTG,AAA,ATC,CAA,AGT,TGT,GGA,GTG,CAG,AAA,TTC,CTA,ATC,TTT,ATA,GAG
24.EMPII-24:Tm=54,60mer
TCA,ATA,AGA,GTT,CCA,TCA,GCA,GTA,TGA,AGT,TCA,ACA,ACT,GCT,CTA,TAA,AGA,TTA,GGA,ATT
25.EMPII-25:Tm=54,60mer
GCT,GAT,GGA,ACT,CTT,ATT,GAA,GCA,GAA,GCA,TGT,GAT,GTT,GGA,TTC,AGA,GAA,GTT,AGA,ATT
26.EMPII-26:Tm=54,60mer
TCA,ACA,ATG,GCT,TTC,CAT,TAA,GAA,GAA,GAA,GAC,CAT,TTT,CAA,TTC,TAA,CTT,CTC,TGA,ATC
27.EMPII-27:Tm=54,60mer
TAA,TGG,AAA,GCC,ATT,GTT,GAT,TAG,AGG,AGT,TAA,TAG,ACA,TGA,ACA,TCA,TCC,TCT,TCA,TGG
28.EMPII-28:Tm=54,60mer
CAA,AAT,ATC,TTG,AAC,CAT,AGT,TTG,TTC,ATC,CAT,AAC,TTG,ACC,ATG,AAG,AGG,ATG,ATG,TTC
29.EMPII-29:Tm=54,60mer
CTA,TGG,TTC,AAG,ATA,TTT,TGT,TGA,TGA,AGC,AAA,ACA,ACT,TCA,ATG,CAG,TTA,GAT,GTT,CTC
30.EMPII-30:Tm=54,60mer
CTA,TCA,CAA,AGA,GTA,TAC,CAA,AGT,GGA,TGG,TTA,GGA,TAA,TGA,GAA,CAT,CTA,ACT,GCA,TTG
31.EMPII-31:Tm=54,60mer
TGG,TAT,ACT,CTT,TGT,GAT,AGA,TAT,GGA,CTT,TAT,GTT,GTT,GAT,GAA,GCT,AAC,ATT,GAA,ACT
32.EMPII-32:Tm=54,60mer
TTG,GAT,CAT,CAG,TCA,ATC,TGT,TCA,TTG,GAA,CCA,TAC,CAT,GAG,TTT,CAA,TGT,TAG,CTT,CAT
33.EMPII-33:Tm=54,60mer
CAG,ATT,GAC,TGA,TGA,TCC,AAG,ATG,GCT,TCC,AGC,AAT,GAG,TGA,AAG,AGT,TAC,TAG,AAT,GGT
34.EMPII-34:Tm=54,60mer
AGA,CCA,AAT,AAT,AAC,ACT,TGG,ATG,GTT,TCT,ATC,TCT,TTG,AAC,CAT,TCT,AGT,AAC,TCT,TTC
35.EMPII-35:Tm=54,60mer
CAA,GTG,TTA,TTA,TTT,GGT,CTT,TGG,GTA,ATG,AAT,CTG,GAC,ATG,GAG,CAA,ATC,ATG,ATG,CAT
36.EMPII-36:Tm=54,60mer
ACT,GGT,CTA,CTA,GGA,TCA,ACA,GAC,TTA,ATC,CAT,CTA,TAC,AAT,GCA,TCA,TGA,TTT,GCT,CCA
37.EMPII-37:Tm=54,60mer
GTT,GAT,CCT,AGT,AGA,CCA,GTT,CAA,TAT,GAA,GGA,GGA,GGA,GCT,GAT,ACT,ACT,GCA,ACT,GAT
38.EMPII-38:Tm=54,60mer
GTT,GAT,CTT,CAT,CAA,CTC,TTG,CAT,ACA,TAG,GAC,AAA,TAA,TAT,CAG,TTG,CAG,TAG,TAT,CAG
39.EMPII-39:Tm=54,60mer
AAG,AGT,TGA,TGA,AGA,TCA,ACC,ATT,CCC,AGC,TGT,TCC,AAA,GTG,GTC,TAT,TAA,GAA,GTG,GCT
40.EMPII-40:Tm=54,60mer
TTC,ACA,CAA,AAT,AAG,TGG,TCT,AGT,TTC,TCC,TGG,AAG,AGA,AAG,CCA,CTT,CTT,AAT,AGA,CCA
41.EMPII-41:Tm=54,60mer
GAC,CAC,TTA,TTT,TGT,GTG,AAT,ATG,CAC,ATG,CAA,TGG,GTA,ACA,GTC,TTG,GAG,GAT,TCG,CAA
42.EMPII-42:Tm=54,60mer
CCT,TGC,AAT,CTA,GGA,TAT,TGT,CTA,AAT,GCT,TGC,CAG,TAC,TTT,GCG,AAT,CCT,CCA,AGA,CTG
43.EMPII-43:Tm=54,60mer
CAA,TAT,CCT,AGA,TTG,CAA,GGA,GGA,TTC,GTT,TGG,GAT,TGG,GTT,GAT,CAA,TCT,CTT,ATT,AAG
44.EMPII-44:Tm=54,60mer
CTC,CTC,CAT,AAG,CAG,ACC,AAG,GGT,TTC,CAT,TTT,CAT,CAT,ACT,TAA,TAA,GAG,ATT,GAT,CAA
45.EMPII-45:Tm=54,60mer
TTG,GTC,TGC,TTA,TGG,AGG,AGA,TTT,CGG,AGA,TAC,TCC,TAA,CGA,TAG,ACA,ATT,CTG,TAT,GAA
46.EMPII-46:Tm=54,60mer
AAG,TGC,TGG,ATG,TGG,AGT,TCT,ATC,AGC,AAA,AAC,AAG,ACC,GTT,CAT,ACA,GAA,TTG,TCT,ATC
47.EMPII-47:Tm=54,60mer
GAA,CTC,CAC,ATC,CAG,CAC,TTA,CTG,AAG,CAA,AAC,ATC,AAC,AAC,AAT,TCT,TCC,AAT,TCA,GAT
48.EMPII-48:Tm=54,60mer
AAA,AGG,TAT,TCA,GAA,GTA,ACT,TCA,ATA,GTT,TGT,CCA,GAC,AAT,CTG,AAT,TGG,AAG,AAT,TGT
49.EMPII-49:Tm=54,60mer
GTT,ACT,TCT,GAA,TAC,CTT,TTC,AGA,CAT,AGT,GAT,AAC,GAA,CTT,CTT,CAT,TGG,ATG,GTT,GCA
50.EMPII-50:Tm=54,60mer
CAA,GAG,GAA,CTT,CAC,CAC,TTG,CCA,ATG,GCT,TAC,CAT,CAA,GTG,CAA,CCA,TCC,AAT,GAA,GAA
51.EMPII-51:Tm=54,60mer
AAG,TGG,TGA,AGT,TCC,TCT,TGA,TGT,TGC,TCC,ACA,AGG,TAA,ACA,ATT,GAT,TGA,ACT,GCC,TGA
52.EMPII-52:Tm=54,60mer
AGT,AAG,CCA,AAG,TTG,ACC,AGC,ACT,TTC,TGG,TTG,TGG,AAG,TTC,AGG,CAG,TTC,AAT,CAA,TTG
53.EMPII-53:Tm=54,60mer
CTG,GTC,AAC,TTT,GGC,TTA,CTG,TTA,GAG,TTG,TTC,AAC,CAA,ACG,CAA,CTG,CAT,GGT,CTG,AAG
54.EMPII-54:Tm=54,60mer
TCT,GCA,AGT,CTC,CAT,TGT,TGC,CAT,GCA,CTA,ATA,TGT,CCT,GCT,TCA,GAC,CAT,GCA,GTT,GCG
55.EMPII-55:Tm=54,60mer
CAA,CAA,TGG,AGA,CTT,GCA,GAA,AAC,CTT,AGT,GTT,ACT,CTT,CCA,GCA,GCA,TCT,CAT,GCA,ATT
56.EMPII-56:Tm=54,60mer
GTT,CAA,TAC,AGA,AAT,CCA,TTT,CAG,AAG,TAG,TAA,GAT,GTG,GAA,TTG,CAT,GAG,ATG,CTG,CTG
57.EMPII-57:Tm=54,60mer
AAT,GGA,TTT,CTG,TAT,TGA,ACT,TGG,TAA,CAA,GAG,ATG,GCA,ATT,CAA,CAG,ACA,ATC,TGG,ATT
58.EMPII-58:Tm=54,60mer
AAG,AAG,TTG,TTT,CTT,ATC,TCC,AAT,CCA,CAT,TTG,AGA,AAG,GAA,TCC,AGA,TTG,TCT,GTT,GAA
59.EMPII-59:Tm=54,60mer
GAG,ATA,AGA,AAC,AAC,TTC,TTA,CTC,CAC,TTA,GAG,ATC,AAT,TCA,CTA,GAG,CAC,CAC,TTG,ATA
60.EMPII-60:Tm=54,60mer
TTA,GGA,TCA,ATT,CTA,GTT,GCT,TCA,CTA,ACT,CCA,ATA,TCG,TTA,TCA,AGT,GGT,GCT,CTA,GTG
61.EMPII-61:Tm=54,60mer
GCA,ACT,AGA,ATT,GAT,CCT,AAC,GCA,TGG,GTT,GAA,AGA,TGG,AAG,GCA,GCA,GGA,CAT,TAC,CAA
62.EMPII-62:Tm=54,60mer
CAA,GAG,TAT,CAG,CAG,TAC,ATT,GCA,ACA,ATG,CTG,CTT,CTG,CTT,GGT,AAT,GTC,CTG,CTG,CCT
63.EMPII-63:Tm=54,60mer
ATG,TAC,TGC,TGA,TAC,TCT,TGC,TGA,TGC,TGT,TCT,TAT,TAC,TAC,TGC,TCA,TGC,TTG,GCA,ACA
64.EMPII-64:Tm=54,60mer
TCT,GTA,AGT,CTT,TCT,ACT,AAT,GAA,CAA,AGT,CTT,ACC,TTG,ATG,TTG,CCA,AGC,ATG,AGC,AGT
65.EMPII-65:Tm=54,60mer
TTA,GTA,GAA,AGA,CTT,ACA,GAA,TTG,ATG,GTA,GTG,GTC,AAA,TGG,CAA,TTA,CTG,TTG,ATG,TTG
66.EMPII-66:Tm=54,60mer
AGT,CCA,ATT,CTT,GCT,GGA,TGT,GGA,GTA,TCA,CTT,GCA,ACT,TCA,ACA,TCA,ACA,GTA,ATT,GCC
67.EMPII-67:Tm=54,60mer
CAT,CCA,GCA,AGA,ATT,GGA,CTT,AAC,TGT,CAA,CTT,GCA,CAA,GTA,GCA,GAA,AGA,GTT,AAC,TGG
68.EMPII-68:Tm=54,60mer
TAA,GTC,TAT,CTG,GAT,AGT,TTT,CTT,GTG,GTC,CCA,ATC,CAA,GCC,AGT,TAA,CTC,TTT,CTG,CTA
69.EMPII-69:Tm=54,60mer
AAA,CTA,TCC,AGA,TAG,ACT,TAC,TGC,TGC,ATG,TTT,CGA,TAG,ATG,GGA,TCT,TCC,ATT,GTC,TGA
70.EMPII-70:Tm=54,40mer
AAA,CGT,ATG,GAG,TGT,ACA,TAT,CAG,ACA,ATG,GAA,GAT,CCC,A
Take above-mentioned EMPII-1 ~ EMPII-70 as primer, utilize the PCR intestinal bacteria lacZ gene that increases.In 50 μ l reaction systems, EMPII-2 ~ EMPII-69 addition of totally 68 primers is 2ng, and the addition of an EMPII-1 and EMPII-702 primer is 30ng, and amplification condition (Protocol) is followed successively by: 94 ℃ of preheating 1min; 94 ℃ of 30s; 50 ℃ of 30s; 72 ℃ of 150s.Totally 35 circulations.
After above-mentioned PCR finishes, 1%(W/V) reclaim after agarose gel electrophoresis, through sequential analysis, the nucleotide sequence of the intestinal bacteria lacZ gene obtaining is as shown in SEQ.ID No.1, by its called after EclacZS gene.
Getting respectively the above-mentioned agarose gel electrophoresis of 10 μ l, to reclaim product directly connected with T/A cloning vector (precious biotechnology (Dalian) company limited, D103A).4 ℃ of connections are spent the night, and after 42 ℃ of heat shocks, Efficient Conversion is in bacillus coli DH 5 alpha competence.
The clone of embodiment 2 clostridium CRO repressor-like DNA-binding gene (OABC) promotors and the structure of plasmid pYN7443
With R27320(5 '-TCT, AGA, AAA, GGA, AAA, TAT, GAT, AAA, AAA, TTT, CA-3 ') and R27321(5 '-GGA, TCC, CAT, TAA, TAT, CGA, AAA, TAG, CTT, AAA, CCC, AAC-3 ') be primer, take the genomic dna (GenBank:AE001437.1) of clostridium as template, carry out the amplification of clostridium OABC gene promoter with the high-fidelity high temperature-resisting DNA polymerase Pyrobest of Japanese TAKARA company.Pcr amplification program is: 94 ℃ of warm start 5min; 94 ℃ of sex change 20S, 53 ℃ of annealing 20S, 72 ℃ are extended 15S, totally 35 circulations; 94 ℃ are extended 10min eventually; PCR product, after 1%Agarose electrophoresis detection, reclaims the fragment of 300bp left and right with the Agarose electrophoresis of same concentration.Reclaim test kit with the UNIQ-10 pillar DNA gel that the raw work in Shanghai is produced and purify pcr amplification product, obtain clostridium OABC gene promoter, its nucleotide sequence is as shown in SEQ ID No.2.Cutting this pcr amplification product with XbaI and BamHI enzyme connects on the pSOS94 carrier that same enzyme cuts, to replace its original ptb gene promoter, obtain pYN7443 plasmid (forms by original ptb gene promoter being replaced with to this clostridium OABC gene promoter structure on the basis of existing carrier pSOS94 method, wherein pSOS94 preparation method is referring to U-B.Tummal et al., 1999, Applied Ana Environmental Microbiology, 9,3793-3799).
The structure of embodiment 3 intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS
Referring to Fig. 1, use respectively BamHI and SacI(purchased from precious biotechnology (Dalian) company limited) carry out double digestion, reclaim DNA fragmentation, by T4DNA ligase enzyme by the EclacZS gene of embodiment 1 be connected-the purifying of pYN7443 plasmid with embodiment 2, obtained the intestinal bacteria-clostridium shuttle vectors pYN7443-EclacZS that contains intestinal bacteria lacZ gene.
The clostridium of embodiment 4 intestinal bacteria-clostridium shuttle expression carrier pYN7443-EclacZS transforms
Clostridium transforms, and concrete operations are as follows:
1) the competent making of clostridium:
Clostridium acetobutylicum after activation is met to (beef peptone l0g/L meat extract 10g/L in bacterium amount access clostridium enriched medium by l%, yeast powder 3g/L, DL-Alanine 3g/L, halfcystine hydrochloric acid 0.5g/L, Zulkovsky starch 1g/L, resazurin 0.03g/L, NaCl3g/L, sodium acetate 3g/L, NaOH adjusts pH=7.0), growth bacterium liquid in the mid-term each 30mL that takes the logarithm respectively, to the vitamins C aqueous solution that adds filtration sterilization in bacterium liquid, its whole mass concentration is 0.03g/mL simultaneously.After bacterium liquid ice bath 10min, 4 ℃ of low temperature, the centrifugal 10min of 4000r/min collect thalline, then use the PEB(0.5mmol/L sucrose of the interpolation vitamins C (whole mass concentration is 0.03g/mL) of precooling, 1mmol/L MgCl
2, the Na of 1mmol/L pH=7.4
3pO
4damping fluid) the solution washing thalline 3 times that suspends, the thalline after washing is resuspended containing ascorbic PEB solution with 200uL, packing immediately for electric transformation experiment or frozen for subsequent use in-80 ℃.
2) electric shock of clostridium transforms:
Get the shuttle expression carrier plasmid (plasmid concentration is about 200ug/uL) that 2uL embodiment 3 obtains, add 100uL clostridium propionicum sense cell, after mixing, place 10IIlin on ice, carry out electrification experiment (voltage 2.5kV, resistance infinity and time 10ms).Cell after transforming is transferred in the anaerobism pipe that 5mL recovery substratum is housed, 37 ℃ of standing recovery 4h, be coated on contain erythromycin and and the flat board of X-gal on, experimental result is referring to Fig. 2, from scheming, can find out that the chief gets up on the flat board that contains erythromycin and X-gal bacterium colony is almost all aobvious blue, illustrate colibacillary lacZ gene can be in clostridium normal expression.
The mensuration of intestinal bacteria lacZ gene in embodiment 5 clostridium positive colonies
1) cultivation of clostridium positive transformant and protein extraction: first in picking embodiment 4 erythromycin and and the flat board of X-gal on cultivate in being placed on anaerobic culture box in aobvious blue positive transformant list colony inoculation and 50ml RCM liquid nutrient medium, then took out 5ml nutrient solution 5 every 3 hours, centrifugal 5min under 000g universal gravity constant, sterile water wash 1 time.The cell precipitation of gained is resuspended with 1mL phosphoroclastic cleavage damping fluid, is then transferred in Eppendorf tube.Use ultrasonoscope cracking bacterium liquid 30min, with the centrifugal 30min of 12,000g, get supernatant and obtain albumen crude enzyme liquid.
2) the beta-galactosidase enzymes enzyme assay of LacZ genes encoding
Obtain carrying out immediately beta-galactosidase enzymes enzyme assay after albumen crude enzyme liquid by aforesaid method, enzyme reaction condition: the substrate ortho-nitrophenyl β-D-galactopyranose (pNPG) that contains 0.8 μ mol in the 0.1mol/L phosphatase reaction buffer system that is 1mL at cumulative volume, containing the albumen crude enzyme liquid that is equivalent to extract in the previous step of 2mg left and right, hatch after 30min by 0.5mol/L sodium carbonate 1mL termination reaction for 37 ℃, can reflect this enzymic activity in the absorbance of 420nm wavelength mensuration with ultraviolet spectrophotometer.The vigor of p-NP that here we define that every milligram of albumen generates 1nM in per minute catalysis is 1U.Experimental result is referring to Fig. 3, can find out that in the increase beta-galactosidase enzymes enzymic activity along with fermentation time, also along with increase, in the time that fermentation time reaches 30h, it is maximum that its activity reaches, and reaches 2436.5U from scheming.
Claims (6)
1. intestinal bacteria lacZ gene, its nucleotide sequence is as shown in SEQ IDNo.1.
2. the promotor of clostridium OABC gene, its nucleotide sequence is as shown in SEQ ID No.2.
3. comprise the pYN7443 plasmid of the promotor of clostridium OABC gene claimed in claim 2.
4. intestinal bacteria-clostridium shuttle expression carrier, is characterized in that, is that the sequence construct that inserts intestinal bacteria lacZ genes encoding claimed in claim 1 between the BamHI of pYN7443 plasmid claimed in claim 3 and SacI restriction enzyme site forms.
5. the construction process of intestinal bacteria-clostridium shuttle expression carrier claimed in claim 4, is characterized in that, comprises the following steps:
1) according to the synthetic intestinal bacteria lacZ gene of clostridium preference codon synthesising biological method;
2) the pYN7443 plasmid of the promotor that structure comprises clostridium OABC gene;
3) described intestinal bacteria lacZ gene is connected with pYN7443 plasmid and builds intestinal bacteria-clostridium shuttle expression carrier.
6. clostridium shuttle expression carrier claimed in claim 4 is transformed in clostridium and measures the method for intestinal bacteria lacZ gene activity, it is characterized in that, comprises the following steps:
1) electric shocking method transforms clostridium;
2) determination of activity of intestinal bacteria lacZ gene.
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| CN114134094A (en) * | 2021-12-07 | 2022-03-04 | 上海市农业科学院 | A construction method of Escherichia coli engineering bacteria for de novo synthesis of riboflavin |
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Non-Patent Citations (3)
| Title |
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| HESHAM ET AL: "Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| JOHN ET AL: "The ClosTron: A universal gene knock-out system for the genus Clostridium", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| LOTHAR ET AL: "Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114134094A (en) * | 2021-12-07 | 2022-03-04 | 上海市农业科学院 | A construction method of Escherichia coli engineering bacteria for de novo synthesis of riboflavin |
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