A method for suppressing horizontal gene transfer
A technology of horizontal transfer and gene inhibition, applied in the fields of molecular biology and genetic engineering, can solve problems such as increasing medical costs and health threats, and achieve the effect of resisting drug-resistant bacteria and reducing transformation efficiency
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Embodiment 1
[0041] 1. PCR amplification of exogenous linear targeting fragment containing kana resistance gene for knocking out loiP gene:
[0042] (1) Using the plasmid pKD4 containing the Kanak resistance gene as a template, use primers K-loiP-F and K-loiP-R to amplify the Kanak resistance gene linear targeting fragment "homologous arm + FRT + Kan Sequence+FRT+homology arm", after the amplification is completed, it is detected by 1% agarose gel electrophoresis.
[0043] The amplification system is shown in Table 1.
[0044] Table 1 PCR system of exogenous kana resistance gene linear targeting fragment
[0045] Reagent name volume 5×prime STAR buffer(Mg 2+ +plus)
10 μL dNTP mixture (2.5mM each) 4μL K-loiP-F 1μL K-loiP-R 1μL template 1μL Primer STAR HS DNA polymerase (2.5units / μL) 0.5μL Sterilized distilled water Up to 50μL
[0046] (2) PCR amplification conditions: pre-denaturation: 95°C, 5min; denaturation: 98°C, 10s...
Embodiment 2
[0071] (1) Loss of the recombinant strain E.coli DH5α::Kan::△loiP Kanna resistance fragment (also can be understood as: deletion or knockout)
[0072] The temperature-sensitive plasmid pCP20, which can encode the recombinant protein FLP, was introduced into the correctly identified E.coliDH5α::Kan::△loiP recombinant strain by electroporation, revived and cultured at 30°C and 150r / min for 1 hour, and 200 μL was spread on the containing 35 μg / mL chloramphenicol on LB plate medium, cultured at 30°C for 8 hours, then increased to 42°C for overnight culture, heat-induced expression of FLP recombinase, and plasmid loss.
[0073] (2) Screening and identification of mutant strain E.coli DH5α::△loiP
[0074]Use a sterile toothpick to randomly pick 5 single colonies grown on the chloramphenicol plate, and inoculate each single colony on the chloramphenicol plate, the ampicillin plate and the plate without antibiotics, and culture at 37°C for 12-16 hours. The colony that can only grow o...
Embodiment 3
[0083] Determination of Transformation Efficiency of Mutant Strain E.coli DH5α::△loiP
[0084] with 100mM CaCl 2 Prepare wild-type E.coli DH5α and mutant strain E.coli DH5α::△loiP competent cells respectively, and dilute plasmids pUC19, pET-32a, p1304 to 5ng / μL. Take 2 μL and add them to 100 μL of the two competent states, mix gently, and ice-bath for 30 minutes. Heat shock at 42°C for 90s, ice-bath for 2min, then add 900μL LB culture medium and re-incubate at 37°C for 50min at 180r / min. Take 100 μL and spread on LB plate respectively, culture overnight at 37°C and count.
[0085] Transformation efficiency = (dilution × number of transformants × volume of transformation stock solution) / volume of coated bacterial solution / DNA mass number (μg)
[0086] The result is as Figure 4-Figure 7 As shown, the results show that the transformation efficiency of the competent cells of wild-type E.coli DH5α and mutant E.coli DH5α::ΔloiP to plasmid pUC19 is 6.29×10 6 CFU / μg and 1.89...
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