Method for evaluating bacterial conjugational transfer efficiency based on quantitative PCR (Polymerase Chain Reaction) technology

A technology of conjugation transfer and technology, applied in the direction of microorganism-based methods, bacteria, recombinant DNA technology, etc., can solve the problems of weak sensitivity, poor repeatability, and low accuracy, and achieve strong sensitivity, high accuracy, and good repeatability Effect

Pending Publication Date: 2022-03-22
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

[0006] Aiming at the problems of weak sensitivity, low accuracy and poor repeatability of the method for evaluating the efficiency of conjugation and transfer by traditional plate screening of bacteria, the present invention proposes a method for evaluating the efficiency of conjugation and transfer of bacteria based on quantitative PCR technology. The suicide plasmid of the homologous sequence of the recipient bacterium; and it is introduced into the donor bacterium Escherichia coli by transformation; then, through the conjugation of the donor bacterium and the recipient bacterium, the suicide plasmid is sent into the recipient bacterium, and combined with the recipient bacterium The homologous sequence on the bacterial chromosome undergoes homologous recombination, and the suicide plasmid is integrated into the recipient cell; according to the chromosomal sequence after homologous recombination, design the specific detection of the upstream primer sequence on the recipient bacterial chromosome and the downstream primer sequence on the plasmid Primers, quantify the number of zygotes by quantitative PCR, and design specific primers for the recipient bacteria (including zygotes and unconjugated recipient bacteria) relative to the donor bacteria, and quantify the total number of recipient bacteria; finally, through the formula : Conjugation efficiency = number of zygotes ÷ total amount of recipient bacteria to obtain conjugation efficiency, where the total amount of recipient bacteria = number of zygotes + number of unconjugated recipient bacteria

Method used

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  • Method for evaluating bacterial conjugational transfer efficiency based on quantitative PCR (Polymerase Chain Reaction) technology
  • Method for evaluating bacterial conjugational transfer efficiency based on quantitative PCR (Polymerase Chain Reaction) technology
  • Method for evaluating bacterial conjugational transfer efficiency based on quantitative PCR (Polymerase Chain Reaction) technology

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Embodiment 1

[0071] Taking the 1161031-1161687 and 1827872-1828471 homologous sequences on chromosome 1 in Vibrio harveylius 345 as an example to illustrate the construction of the bacterial conjugative transfer efficiency evaluation system by quantitative PCR ( figure 1 ).

[0072] The 1161031-1161687 sequence on chromosome 1 in Vibrio harveylius 345, with a length of 657bp, is located between the CU052_05900 gene and the CU052_05905 gene, belonging to the intergenic region; the 1827872-1828471 sequence on chromosome 1, with a length of 600bp, includes CU052_092703' end 128bp, CU052_09270 gene and the intergenic region of the CU052_09275 gene and the 151 bp of the 3' end of the CU052_092755.

[0073]In this study, the bacterial conjugative transfer efficiency evaluation system based on the quantitative PCR method of the homologous sequences of 1161031-1161687 and 1827872-1828471 on chromosome 1 in Vibrio harvelii 345 will be the key regulatory genes and influencing factors of horizontal g...

Embodiment 2

[0129] The bacterial conjugative transfer efficiency evaluation system based on the quantitative PCR method based on the homologous sequence of 1827872-1828471 on chromosome 1 in Vibrio harveylius 345 was used to analyze the logarithmic phase of Vibrio harveylius 345 and its gene knockout strain 345ΔCU052_28220 (GenBank number is AWB03109. 1) Conjugative transfer efficiency. Specific steps are as follows:

[0130] (1) Streak the donor bacterium pSW7848-HS2-E.coliGEB883 on an LB solid plate supplemented with 20 μg / mL chloramphenicol and 0.3 mMDAP (2,6-Diaminopimelic acid), and culture it overnight at 37°C for activation;

[0131] (2) Pick 3 monoclonal colonies formed by the activated bacteria in (1), inoculate them in LB liquid medium supplemented with 20 μg / mL chloramphenicol and 0.3 mM DAP, and cultivate to logarithm at 37°C and 200 rpm Early OD600nm=0.3~0.7;

[0132] (3) Configure an LBS solid plate, and activate the recipient bacteria Vibrio harvey 345 and 345ΔCU052_28220...

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Abstract

The invention discloses a method for evaluating bacterial conjugational transfer efficiency based on a quantitative PCR technology, and belongs to the technical field of bacterial molecular biology. The method comprises the following steps: constructing a suicide plasmid containing a recipient bacterium homologous sequence through PCR (Polymerase Chain Reaction) amplification and seamless cloning; introducing the strain into donor bacteria escherichia coli in a transformation mode; sending the suicide plasmids into recipient bacteria under the joint action of the donor bacteria and the recipient bacteria, carrying out homologous recombination on the suicide plasmids and homologous sequences on chromosomes of the recipient bacteria, and integrating the suicide plasmids into recipient cells; according to the chromosome sequence after homologous recombination, a specific detection primer with an upstream primer sequence on a recipient bacterium chromosome and a downstream primer sequence on a plasmid is designed, the number of zygotes is quantified in a quantitative PCR mode, meanwhile, a specific primer of recipient bacteria relative to donor bacteria is designed, and the total number of the recipient bacteria is quantified; and finally, obtaining the jointing efficiency according to a formula: jointing efficiency = number of the zygotes / total amount of the recipient bacteria.

Description

technical field [0001] The invention relates to the technical field of bacterial molecular biology, in particular to a method for evaluating bacterial conjugation transfer efficiency based on quantitative PCR technology. Background technique [0002] Horizontal gene transfer (HGT) is different from vertical gene transfer, and is the exchange of genetic material between different individuals within or between species. There are three main forms of horizontal gene transfer in prokaryotes: transformation, transduction, and conjugative transfer. Among them, bacterial conjugative transfer is the most common method of horizontal gene transfer, which builds a conjugative bridge between the donor cell and the recipient cell to form physical contact, thereby transferring DNA from the donor to the recipient. [0003] Exogenous DNA enters recipient bacteria through horizontal gene transfer (HGT), a process that allows genetic material to rapidly diffuse among individuals of different ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12N15/11C12N15/74C12N1/21C12R1/63
CPCC12Q1/689C12Q1/6851C12N15/74C12N1/20C12Q2531/113C12Q2537/165
Inventor 邓益琴司徒洁金冯娟陈皓祥
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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