Isolation of Otsba Gene Involved in Trehalose Synthesis

The present study was carried out to isolate otsBA gene involved in trehalose synthesis from metagenome of sand dunes of Bikaner. Isolated metagenomic DNA was amplified using two different sets of primers homologous to E. coli, one designed through Primer Premier Software of NCBI, could not generate any specific fragment. Simultaneously, amplification was also carried out using primer pair as per Padilla et al., (2003). However most amplification generated by both the pair were very large with some smear. Commercially supplied pGEM-T Easy vector system was used for cloning of amplified product through A/T cloning. Transformation frequency was found maximum with 1:12 vector: insert molar ratio as compared to 1:4 and 1:8, ligated DNA. Transformation efficiency was found to be 1.38 X 104/µg of DNA. Clones developed were between the size range of o.5kb to 2kb and none was found to improve osmotic stress tolerance in DH5? when transformed.