KLONING GEN carB Salmonella typhi MENGGUNAKAN VEKTOR EKSPRESI pET-16b dan SEL INANG Escherichia coli JM109

Abstract

The effort of cloning the Lister strain of Salmonella typhi (NCTC 786, BCC 712) carB gene using pET-16b expression vector and E. coli JM109 as host cell has been done. The carB gene and the pET-16b expression vector were both prepared from their recombinant plasmid digested using BamHI and NdeI as restriction enzymes. The pG-carB-11-ST recombinant plasmid was isolated from Escherichia coli XL10(pG-carB-11-ST) and the pET-carA-ST recombinant plasmid was from Escherichia coli DH5α(pET-carA-ST). After being ligated (in a ratio of vector:gene of 1:3, 1:5 and 1:6) in the presence of T4 Bacteriofage DNA Ligase, the ligation mixture was used to transform Escherichia coli JM109 cells and plated out onto Luria Bertani medium containing ampicillin. An amount of 369 produced colonies were screened for the presence of the appropriate recombinant plasmid using combination of plasmid miniprep and agarose gel electrophoresis. None of the recombinant plasmids being suspected to carry the Salmonella typhi carB gene. It is suggested to repeat cloning process using E. coli JM109 or using E. coli XL10 as host cell which were known to have large cloning efficiency and can be used for large plasmids up to 25 kb.