Traceable non-fluorescence-marked escherichia coli and preparation method thereof
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A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of bioengineering, can solve problems such as uncontrollable insertion sites, achieve the effect of convenient tracking and traceability, and reduce impact
Inactive Publication Date: 2016-03-23
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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However, during the insertion process of this method, due to the randomness of gene insertion mediated by transposons, the insertion site is often uncontrollable, so specific site-directed gene insertion technology is required, mostly with the help of λ phage recombinase (Red system) Realized, they are coded by gam, bet and exo
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[0020] The embodiment of the present invention provides a method for preparing traceable non-fluorescence-labeled Escherichia coli. For the schematic diagram of the method, see figure 1 , including the following steps:
[0021] S10, using pKD4 as a template, the FRTcassette product containing a sequence homologous to the manX gene is amplified by PCR with pKD4 amplification primers;
[0022] S20, transforming the pKD78 plasmid into Escherichia coli to be transformed, and obtaining Escherichia coli containing the pKD78 plasmid;
[0023] S30, transforming the FRTcassette product into Escherichia coli containing the pKD78 plasmid and performing site-directed recombination with the chromosome of Escherichia coli to form recombinant Escherichia coli;
[0024] S40, the kanamycin resistance gene in the recombinant E. coli chromosome FRTcassette is removed by the suicide plasmid pCP20, and the traceable non-fluorescence-labeled E. coli of the present invention is obtained.
[0025] ...
Embodiment 1
[0041] S10, using pKD4 as a template, using the above-mentioned upstream primer HP1 and downstream primer HP2 to amplify to obtain a FRTcassette product with a size of about 1600 bp containing a sequence homologous to the Escherichia coli manX gene.
[0042] Specific PCR reaction system (50μl):
[0043]
[0044] PCR process:
[0045]
[0046]
[0047] After the PCR process, the FRTcassette product was purified and recovered by 1% agarose gel electrophoresis for future use.
[0048] S20, transforming the pKD78 plasmid into Escherichia coli to be transformed to obtain Escherichia coli containing the pKD78 plasmid, the detailed implementation steps include:
[0049] S21, preparing competent E. coli, the specific method steps can be carried out with reference to the steps introduced in the "Molecular Cloning Experiment Guide" (third edition); however, the cell suspension in the one-step method adopts the following components specifically developed by the present inventio...
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Abstract
The invention provides a preparation method of traceable non-fluorescence-marked escherichia coli. The method comprises the steps that an FRT cassette product which contains the homologous sequence with a manX gene of escherichia coli to be transformed is obtained through PCR amplification by taking pKD4 as a template; pKD78 plasmids are transferred into the escherichia coli to be transformed to obtain escherichia coli containing the pKD78 plasmids; the FRT cassette product is transferred into the escherichia coli containing the pKD78 plasmids, and site-specific recombination is performed to form recombined escherichia coli; a kanamycin resistance gene in a chromosome FRT cassette of the recombined escherichia coli is knocked out through suicide plasmids pCP20. According to the method, only an FRT trace with nucleotides not more than 50 is finally left at the specified position in a genome of the transformed escherichia coli, the influence of insertion sequences on a host bacterium is greatly reduced, resistance characteristic propagation caused due to the fact that the transformed escherichia coli is implanted the host cannot changed, and the FRT trace can facilitate follow-up tracing of the transformed strain.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a traceable non-fluorescent labeled Escherichia coli and a preparation method thereof. Background technique [0002] Escherichia coli (Escherichia coli, E.coli) as a model organism, an important aspect of it is that it can be transformed as a host bacterium to produce various useful products, such as fuels, proteins and polymers. In the early E. coli transformation, for example, there were non-essential gene knockout technology, high-efficiency expression technology of endogenous or exogenous genes, etc. Among them, the high-efficiency expression technology of endogenous or exogenous genes mostly relies on the tool of plasmid, through which the target gene is recombined and introduced into expression, which has the advantages of convenient operation and controllable expression. However, on the one hand, due to the copy number and various other DNAs carried by ...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
Inventor 陈声郭九标林大川
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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