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Method for knocking out gene in clostridium acetobutylicum

A clostridia and gene technology, applied in the field of genetic engineering, can solve the problems of low homologous recombination efficiency, difficulty in obtaining double-crossover transformants, etc.

Inactive Publication Date: 2012-07-11
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Based on these problems, the inventors tried to establish a new knockout technology in Clostridium acetobutylicum, using the expression of I-SceI homing endonuclease to overcome the low efficiency of homologous recombination in this bacterium, and it is difficult to obtain double The problem of exchanging transformants, and can complete gene knockout without trace

Method used

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  • Method for knocking out gene in clostridium acetobutylicum
  • Method for knocking out gene in clostridium acetobutylicum
  • Method for knocking out gene in clostridium acetobutylicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1, the construction of pUC19-adc plasmid vector

[0098] 1. Construction of pUC19-adc plasmid vector

[0099] Using the Clostridium acetobutylicum genome as a template, primers HindIII (adc-L1) and R1-L1 (adc) were used to amplify adc upstream homology arm adcL by PCR; primers L2-R2 (adc) and BamHI (adc- R2) Amplify the downstream homology arm adcR of adc by PCR; after the PCR product is recovered (see the Huashun Glue Recovery Kit for the method), it is digested with BamHI and HindIII and ligated into the same digested plasmid pUC19 (see the Huasun Glue Recovery Kit for the method) ; Use T4 ligase (Takara Company) to ligate the digested fragment and the vector, and then overnight at 16°C. The resulting plasmid was named pUC19-adc.

[0100] Use pUC19-adc to transform E.coli DH5α competent cells, see Molecular Cloning 3 for the method. Then the recombinants were picked, cultured in LB test tubes (Amp) at 37°C overnight, the plasmids were extracted, and iden...

Embodiment 2

[0109] Embodiment 2, the construction of pSY9-1-adc plasmid vector

[0110] Using the Clostridium acetobutylicum genome as a template, primers NdeI (SceI-thl1), Eml-Thl1 were used to amplify the thl promoter (including the rbs sequence) by PCR, and introduced the I-SceI homing enzyme recognition site 18bp; at the same time Using the pIMP1 plasmid as a template, using Thl2-Em2 and NdeI (XholI-Em-2) as primers, PCR amplifies the Em resistance gene, respectively purifying the PCR products, using it as a template, and using NdeI (SceI-thl1), NdeI (SceI-thl1), NdeI (XholI-Em-2) was used as a primer, and the sequence containing thl promoter + RBS + MLS was obtained by Overlapping PCR. After digestion with NdeI, it was connected into the pUC19-adc plasmid with the same restriction enzyme digestion. Enzyme digestion identification chart see Figure 13 ), select the forward transformant, named as pSY9-1-adc (see figure 1 and SEQ ID NO: 2).

[0111] The primer sequences used are as f...

Embodiment 3

[0117] Embodiment 3, the construction of pSY14-thl plasmid vector

[0118] Due to the large gap between the original sequence of I-SceI and the use of codons in Clostridium, the inventors optimized the codons of the gene, which was synthesized by Nanjing Jinsiruiji Biotechnology Co., Ltd., and the synthesized gene was cloned in a commercially available The pUC57 plasmid is named: pUC57-SceC. The sceC sequence (SEQ ID NO: 1) was amplified by PCR with primer SceC-1 (BamHI) and primer SceC-2 (SmaI). After double digestion with BamHI and SmaI, it was connected into the pITC plasmid digested with the same enzyme. After enzyme digestion verification (for the enzyme digestion identification diagram, see Figure 14 ), named pSY14-thl (see figure 2 and SEQ ID NO: 3).

[0119] The primer sequences used are as follows:

[0120] SceC-1 (BamHI): 5`-CGCGGATCCATGCATCAAAAGAATCAAGT-3` (SEQ ID NO: 12)

[0121] SceC-2 (SmaI): 5`-TCCCCCGGGTTATTTTAAGAAAAGTTTCAC-3` (SEQ ID NO: 13)

[0122] Oth...

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PUM

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Abstract

The invention relates to a method for realizing gene knock out, exogenous gene introduction, point mutation and large fragment knock out in clostridium, polynucleotide sequences containing a sequence shown by SEQ ID NO: 1, constructions and the like which are used in the method, and the application of the polynucleotide sequences and the constructions to realization of the gene knock out, the exogenous gene introduction, the point mutation and the large fragment knock out in the clostridium. The invention also relates to the clostridium prepared by the adoption of the method.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to two recombinant plasmids, pSY9-1-adc and pSY14-thl, which can be used for gene knockout of Clostridium acetobutylicum. Background technique [0002] Due to the limitation of oil resources and the volatility of international crude oil prices, bio-butanol is currently receiving more and more attention. Clostridium acetobylicum (Clostridium acetobylicum) is a Gram-positive, spore-forming, obligate anaerobic bacterium that can utilize a variety of carbon sources and produce solvents acetone, butanol, and ethanol during fermentation. Therefore, Clostridium acetobutylicum has also received widespread attention from researchers from various countries. In recent years, there have been more and more metabolic engineering studies on Clostridium acetobutylicum. 1-3 . [0003] However, the bacterium has always had the problem of lack of a genetic operating system. B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63C12N15/09C12N15/74C12N1/21
Inventor 邵丽君杨晟姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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