Intrusive lactic acid bacterium and preparation method thereof
A lactic acid bacteria, lactic acid technology, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve problems such as weak ability to deliver DNA, no invasive ability, etc.
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Embodiment 1
[0022] Example 1 PCR amplification of target genes pgsA', Rck-FLAG and mRck-FLAG
[0023] 1. Main experimental materials and molecular biology reagents
[0024] (1) Strains and plasmid vectors
[0025] Escherichia coli-lactic acid bacteria shuttle plasmid vector pW425et, Lactobacillus acidophilus CICC6075, Lactobacillus plantarum NC8 strain, and Salmonella typhimurium χ3761 were all preserved by our laboratory.
[0026] (2) Main materials and reagents
[0027] Bacterial Genome Extraction Kit, Common Plasmid Mini-Extraction Kit, and DNA Gel Recovery Kit were all purchased from OMEGA; high-fidelity PCR polymerase, restriction endonuclease, T4DNA ligase, DL2000 marker, DL5000 marker, λ-hindIII marker and 4×SDS buffer were provided by Takara; ampicillin and erythromycin were packaged by Amresco; CloneJET PCR Cloning Kit was purchased from Thermo; electro-cups were provided by Bio-Rad; nitrocellulose membrane (NC membrane) was purchased from Whatman Company; Anti-FLAG antibody (...
Embodiment 2
[0057] Example 2 Ligation of target gene after enzyme digestion and expression vector
[0058] 1. Double digestion of pW425et vector
[0059] a. The enzyme digestion system is as follows, digest in a 37°C water bath for 2 hours:
[0060]
[0061] b. Use the gel recovery kit to recover and purify the digested product, and detect it by 0.8% agarose gel electrophoresis. pgsA' For the PCR results of the gene, see Figure 4 .
[0062] 2. Construct pW425et-Rck-FLAG and pW425et-pgsA ， -mRck-FLAG recombinant plasmid
[0063] a. The connection system between the target gene and pW425et is as follows:
[0064]
[0065] Connect overnight at 16°C on a ligation instrument, transform into E.coli TOP10 chemically competent cells the next day, spread LB / Em (20 μg / ml) plates, and culture overnight at 37°C until monoclonal formation. Pick 2 clones randomly from each plate, add 5mL LB liquid culture solution (50μl Em) respectively, put them in a shaker, 37°C, 180rpm for 10-12h, use ...
Embodiment 3
[0068] Example 3 Electric transformation of the recombinant plasmid into Lactobacillus acidophilus
[0069] (1) Take 5 μl of recombinant plasmids pW425et-Rck-FLAG and pW425et-pgsA respectively ， Add -mRck-FLAG to two tubes of 100μl Lactobacillus acidophilus competent in ice bath one by one, mix gently, transfer to the pre-cooled electric shock cup with a distance of 0.2 cm, let it stand in the ice bath for 5 minutes, and then put it into the electrotransformer Electric shock in (2.5kV, 6ms);
[0070] (2) Immediately after electroporation, put it in an ice bath for 5 minutes, take the liquid in the electroshock cup and add it to 800 μl 30℃ preheated MRS culture medium (containing 0.5mol / L sucrose), and incubate under anaerobic conditions at 30℃ for 3 hours;
[0071] (3) Evenly spread 100μl of the above bacterial solution on the MRS solid medium containing Em (20mg / ml), and cultivate under anaerobic conditions at 30°C until a single colony in good condition grows, about 18-24h....
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