The Modification Of The Expression Elements Of LipA Gene In Bacillus Subtilis

In genentic engineering of microorganisms, using promoter and stabilizer will be able to achieve a high level of gene expression. In this paper, the A1 promoter of phageΦ29 and the stabilizer of aprE gene of B.subtilis DB104 were chosen to form a new expression box, in order to replace all the expression element of lipA gene of B.subtilis DB104 in site.Recombinant fragment of LE, which is consisted of homologous segments of lipA gene and ermC fragment, was constructed by over-lap PCR; and then the recombinant plasmid of pT1-LE, with the method of state transformation to B.subtilis DB104, an erythromycin resistance strain called DB104E was constructed with its lipA gene delition. Recombinant plasmid of pT1-LA with wild type lipA segments was constructed, and then transformed to DB104E to restored lipA gene; it found that DB104 growed well on olive minimal medium, but DB104E didn't grow at all, which shows that olive minimal medium can be used to screen the transformation of lipA~+. Next recombinant fragment LPAS which contain a new expression element was constructed by over-lap PCR, then the recombinant plasmid of pT1-PAs; the result of sequencing proved that the A1 promoter of phageΦ29 and the stabilizer of aprE gene were connected as designed. However, there was no single colony on olive minimal medium after several times of transforming to DB104E strain using pT1-PAs. It is presumed that this new expression box may have no function or problems may exist in transformation. To prove double exchange genetic recombination really happen, we designed to insert the chloramphenicol resistance gene to the EcoRI site upstreamΑ1 promoter in pT1-PAs by digestion and ligase; however, it had no result within about 70 strains because of lacking marks and nonstandard reaction system. In addition, primers design are limited by particular sequence, the new expression box, chloramphenicol resistance gene and homologous sequences, did not connect together. However, according to the experience obtained, this method of modifying the expression element of lipA gene on chromosome in situ is feasible.