Page 148 - Demo

152 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYantibiotics whenever necessary (ampicillin up to100 mg ml-1) for the selection or maintenance of plasmid. pGEM-T easy system I (Promega, USA) and pET-32a(+) (Stratagene, USA) were used as vectors for cloning of polymerase chain reaction (PCR) products and expressing genes respectively. The pNeo32 cosmid containing partial gene cluster, those sequence analysis were putatively involved in methyl aminosugar biosynthesis pathway, was used as a template for the amplification of ncsS53.2.2. Gene cloning and creation of expression vectors. DNA preparation, its manipulations, restriction endonuclease digestion and DNA ligation were carried out following the standard protocol [8]. Computer aided data base searching and sequence analysis were carried out using BLAST server (http://www.ncbi.nlm.nih.gov) [9]. A set of primers ncsS2-F (5%u2019-GGA GAATTC ATGGCCGGACT CGTAGCG-3%u2019) and ncsS2-R (TGAAGCTTCCGCGTCCCCC TCAGCGGT) (the underline and bold letters indicate the restriction sites) was used for the amplification of nucleotide sequences of ncs53 and the PCR product (999 bps) was cloned into the KpnI and HindIII site of pET-32a (+) to generate pncsS vector. The PCR products were cloned into pGEM-T vector (Promega, USA) and sequenced prior to cloning into the expression vectors to verify that no mutation had been introduced during the PCR amplification. PCR was performed in a thermocycler (Takara, Japan) under the following conditions: 25 cycles of 30 s at 95 %u00b0C, 1 min at 55 %u00b0C and 1 min at 72 %u00b0C. The recombinant expression vectors were introduced into E. coli BL21(DE3) by heat pulse transformation [13].