Recombinant plasmid for acetone-butanol clostridium gene disruption

A technology of Clostridium acetobutylicum and recombinant plasmid, applied in the field of genetic engineering, can solve the problems of inconvenient basic and applied research, imperfect genetic operating system, etc., and achieve the effect of improving insertion efficiency

Inactive Publication Date: 2008-12-24
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Due to the imperfection of the existing genetic operating system of Clostridium acetobutylicum, it is inconvenient for basic and applied research. Therefore, a new genetic operating sy

Method used

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  • Recombinant plasmid for acetone-butanol clostridium gene disruption
  • Recombinant plasmid for acetone-butanol clostridium gene disruption
  • Recombinant plasmid for acetone-butanol clostridium gene disruption

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 , Construction of pSY6 plasmid vector

[0047] Using the pJIR750ai plasmid as a template, amplify the L1.LtrB class II intron and the IEP fragment (about 3.4kb in length) encoded by the ltrA gene contained in the plasmid by PCR, digest the amplified product with NdeI, and ligate it into The recombinant plasmid vector obtained from the E.coli-C.acetobutylicum shuttle vector pIMP1-ptb that was also digested and dephosphorylated was named pSY6. The specific experimental steps are as follows:

[0048] 1.1. Primer design and synthesis

[0049] Primer pair In-1 (SEQ ID NO: 4) and In-2 (SEQ ID NO: 5) were designed, NdeI and XhoI restriction sites were introduced at the 5' end of primer In-1, and restriction sites of NdeI and XhoI were introduced at the 5' end of primer In-2. An NdeI restriction site was introduced at the 5' end, where NdeI was used to construct pSY6, while XhoI facilitated the connection of subsequent targetron knockout fragments.

[0050] 1.2. P...

Embodiment 2

[0064] Example 2 , Construction of pSY6-buk, pSY6-solR plasmid vector

[0065] The buk targetron fragment and the solR targetron fragment were respectively amplified by PCR, digested with XhoI and BsrG I, and respectively ligated with the same digested pSY6 vector to obtain knockout plasmids pSY6-buk and pSY6-solR. Among them, the template and primer design of amplifying buk targetron and solR targetron are derived from the Targetron of Sigma-Aldrich Company TM Gene Knockout System (TA0100) Kit. Specific steps are as follows:

[0066] 2.1. Primer design and synthesis

[0067] ReferenceTargetron TM The method provided by the Gene Knockout System (TA0100) Kit designed primers buk-IBS (SEQ ID NO: 6), buk-EBS1d (SEQ ID NO: 7) and buk-EBS2 (SEQ ID NO: 8) respectively for constructing pSY6-buk plasmid vector; and solR-IBS (SEQ ID NO: 9), solR-EBS1d (SEQ ID NO: 10) and solR-EBS2 (SEQ ID NO: 11) were used to construct pSY6-solR plasmid vector.

[0068] In addition, it is also ...

Embodiment 3

[0083] Example 3 , Construct the recombinant strain Clostridium acetobutylicum of buk gene knockout

[0084] After the pSY6-buk plasmid was methylated at the Cac8 I site by E.coli ER2275 / pANS, it was electroporated into Clostridiumacetobutylicum ATCC 824. After recovery overnight, 100 μl of the cell solution was spread on the Amp (25 μg / ml) resistance plate, and the ℃, after 48-96 hours of cultivation in the anaerobic box, the colony PCR is used to verify the single bacteria, and the specific steps are as follows:

[0085] 3.1. Methylation of pSY6-buk plasmid

[0086] E.coli ER2275 was transformed with pANS to obtain E.coli ER2275 / pANS.

[0087] Prepare chemically competent cells of E.coli ER2275 / pANS, and then transform the pSY6-buk plasmid into E.coliER2275 / pANS. Since the pANS plasmid has spectinomycin resistance, it contains 100μg / ml Amp, 50μg / ml spectinomycin Cultivate overnight on the LB medium plate, and then pick a single colony to 4ml LB medium added with 100 μg / m...

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Abstract

The invention provides a recombinant plasmid, a preparation method thereof, an application, two knockout recombinant plasmid vectors pSY6-buk and pSY6-solR which are prepared by taking the recombinant plasmid as a starting plasmid, a method for preparing the recombiant plasmid vectors pSY6-buk and pSY6-solR, and an application. The recombinant plasmid can be taken as a knonckout starting plasmid of an acetone and butanol, can perform the gene knockout efficiently and rapidly, and can be evolved into a novel genetic manipulation system in order to improve the imperfect condition of the prior manipulation system in the current research on the clostridium acetobutylicum, thereby providing convenience for the research on the metabolic engineering of the clostridium acetobutylicum. The recombinant plasmid vectors pSY6-buk and pSY6-solR can be directly applied to establishing knockout strains of the clostridium acetobutylicum buk and solR genes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant plasmid pSY6 which can be used for gene interruption of Clostridium acetobutylicum. Background technique [0002] Clostridium acetobylicum (Clostridium acetobylicum) is a Gram-positive, spore-forming, obligate anaerobic bacterium that can utilize a variety of carbon sources to produce the solvents acetone, butanol, and ethanol (Abou- Zeid, A.A., Fouad, M., and Yassein, M. Zentralbl Bakteriol Naturwiss 1978. 133:125-134). Acetone and butanol are bulk basic raw materials with multiple uses. They can be used as precursors for the synthesis of various organic compounds in the chemical and chemical fields such as dyes, paints, plastics, resins, and rubbers; they are used in the production of antibiotics and synthetic drugs. An essential solvent; it is also a food-grade extractant in the food and spice industries. On the other hand, butanol is st...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 杨晨邵丽君胡世元顾阳陈军杨蕴刘姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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