The Influence Of 5’-UTR To STM542 Strains FlhDC Gene Expre- Ssion And The FlgK Gene Of E. Coli TOP10 Knock Out

Bacterial flagellum is beneficial to bacteria survival which is the result of adapting to the environment in the long process of evolution. Bacteria can escape adverse environment and move toward favorable environments throμgh flagella driving. Bacterial flagellum has chemotaxis which can draw on advantages and avoid disadvantages. The transcription of bacterial flagella gene is all start from the flhDC operon. FlhDC operon includes two genes:flhD and flhC gene. Therefore, flagellum generation is determined by the expression of flhDC gene. This study preliminary cloned the different length of 5’-UTR flhDC gene, the length respectively is 986bp, 1201bp,1572bp,1752bp and 1938bp. Then connecting them to the pBAD33 vector and tansforming into salmonella strain STM542. Finally, evaluating promoter activity of different length segments thoμgh the size of colony diameter on the motility plate. The results showed the size of colonies which are corresponding to 1752bp and 1938bp fragment transformant is approximate. They are all bigger than col one which is corresponding to 1201bp fragment transformant, while the colone which is corresponding to 1572bp fragment transformant is smallest.In order to measure promoter activity of different length of 5’- UTR flhDC gene, this study constructed T vector which regard lacZ as report gene, then connecting promoter fragment of different length of 5’- UTR flhDC gene to the T vector which has report gene and tansforming into escherichia. Finally, evaluating promoter activity of different length segments thoμgh measuring β-Galactosidase activity. The results showed 1752bp and 1938bp fragment promoter activity is approximate.1201bp fragment promoter activity is smallest, while 1572bp fragment promoter activity is the biggest. The size of colony which is corresponding to 1572bp fragment transformant is smallest on motility plate(contradiction in apparent), speculating that promoter whose activity is very strong probably inhibit the division of bacteria.Application Red recombination combining selection and counterselectioncan konck out and konck genome scarelessly. Compared with streptomycin resistance genes(strR), streptomycin sensitive gene(strS) is the dominant phenotypes, therefore strs is a potential reverse screening gene. This study increased the expression of rpsl gene thoμgh constructing pAra-StrS-kanR seletion kit solving the problem that TOP10 was still streptomycin resistance, when rpsL gene was in one copy.