The overall goal of this procedure is to make genome wide single gene deletions in streptococcus sanguine by high throughput PCR. This is accomplished by first obtaining three amplicons by high throughput PCR amplification, including the antibiotic resistance gene, as well as the upstream and the downstream DNA sequences of the target gene. The second step is to obtain the linear recombinant PCR amplicon using three PCR amplicons as templates.
Next, the competent s sanguine cells are prepared. The final step is to transform the linear recombinant PCR amplicon into competent s sanguineous cells. Ultimately colon A PCR amplification and DNA sequencing are used to verify the correct mutants.
The main advantage of this technique over exhausted other method like suicide plasmid knockout, is this method can create the genome wide deletion mutant directly use high throughput. PCR Primers can be designed and synthesized as described in the written protocol using in-house scripts based on the streptococcus sanguineous SK 36 genome sequence. Three sets of primers were designed for amplification of the one kilobase upstream sequence of the target gene, the A PHA three gene ENC coding, kanamycin