This protocol can be used for inactivating chromosomal genes to isolate mutants for gene function study and bacteria. It makes the homologous recombination process simple, fast, and highly efficient. Using this technique, the virulence related genes of the pathogen can be deleted to obtain the attenuated mutant which can be used as a candidate for the attenuated vaccine.
Begin by amplifying the chloramphenicol cassette containing hemology fragments of the micC gene. Design forward and reverse primers to amplify the chloramphenicol cassette from plasmid PKD3, including 50 base pair homology extensions from the five prime and three prime ends of the micC gene. Perform PCR as described in the text manuscript using the PKD3 plasmid as the template to amplify the chloramphenicol cassette.
Determine the size of the PCR product with agarose gel electrophoresis. Then purify the product with a DNA gel recovery kit and determine the concentration of DNA by spectrophotometry. Carry out a second PCR reaction to eliminate the interference of further recombination by the PKD3 plasmid.
Dilute the first PCR product at a ratio of 1 to 200 and use it as a template for the secondary PCR. To construct the first recombinant strain 50336 delta micC dot cat, mix 100 microliters of SE 50336 competent cells with five microliters of PKD 46 plasmid and incubate on ice for 30 minutes. Heat shock the mixture at 42 degrees Celsius for 90 seconds, and rapidly transfer it back on ice for two minutes to transform the plasmid into the cells.
Screen positive colonies by culturing the cells overnight at 30 degrees Celsius on an ampicillin resistant plate. The next day, add 30 milli more L arabinose to the SE 50336 PKD 46 liquid culture and induce recombinase expression with a one hour incubation at 30 degrees Celsius while shaking. Mix 100 nanograms of the purified PCR product and 40 microliters of SE 50336 PKD 46 competent cells in an electric shock cup.
Then carry out electric shock transformation. After electro transformation, transfer the mixture to one milliliter of SOC medium and a shaking culture incubator at 150 RPM and 30 degrees Celsius for one hour. Spread the mixture on a chloramphenicol resistant LB plate and culture at 37 degrees Celsius overnight to screen for positive colonies.
Culture the positive colonies at 42 degrees Celsius for two hours. Then screen the colony for sensitivity to ampicillin and resistance to chloramphenicol at 37 degrees Celsius overnight to obtain the first recombinant strain without PKD 46. After extracting the 50336 Delta micC dot cat genomic DNA using PCR and gel electrophoresis, electroplate 100 nanograms of plasmid PCP 20 into 40 microliters of 50336 Delta micC dot cat competent cells.
Screen positive transformants on both ampicillin and chloramphenicol resistant plates at 30 degrees Celsius. Transfer the positive transformants into non-resistant LB liquid medium and culture them overnight at 42 degrees Celsius. Then isolate single colonies on an LB plate at 37 degrees Celsius.
Select the colony that is sensitive to both ampicillin and chloramphenicol. This mutant is the micC deletion mutant SE 50336 Delta micC. Verify 50336 Delta micC by performing PCR as described in the text manuscript.
This protocol was used to construct the deletion mutant 50336 Delta micC in the complimented mutant 50336 Delta micC, pmicC. The first recombinant 50336 Delta micC dot cat was validated by PCR with an expected band size of about 1200 base pairs of PCR products with the chloramphenicol insertion compared to 279 base pairs in the wild type strain. In the second recombination, the chloramphenicol cassette was eliminated by PCP 20.
The PCR results combined with sequencing confirmed that the isogenic micC mutant was constructed successfully. The expression of ompA, ompC, and ompD genes in strains 50336, 50336 Delta micC, and 50336 delta micC pmicC were analyzed by realtime quantitative PCR. Transcription of ompA and ompC and 50336 delta micC increased about 2.2 fold and threefold compared to the wild type strain.
While transcription of ompD only increased slightly. LD 50 assays showed that after infecting six to eight week old BALB/c mice with each of the three strains, most mice infected by 50336 Delta micC displayed lassitude, inappetence, or diarrhea 48 hours post-infection and died after 96 hours. The mice infected by wild type strain and 50336 delta micC pmicC displayed the same symptoms 72 hours post infection and died after 120 hours.
The LD 50s of the three strains indicated that the virulence of the mutant 50336 Delta micC enhanced 2.5 fold as compared to the wild type. This technique helps researchers study the function of bacterial genes and obtain the desired engineered strain by deleting target genes.
