Degenerate primer design for the clone of acetate kinase of the biohydrogen-producing bacteria B49 using CODEHOP software

This work used CODEHOP to design the degenerate primers of acetate kinase, chose one pair of degenerate primers named ACKJ1 and used the high-biohydrogen bacteria B49 genome DNA as template to make degenerate PCR. 655bp PCR product was obtained, which was transformed into the E. coli DH5α through being linked with pMD18-T vector and sequenced after filtration. Similarity alignment showed that the products of the cloned DNA were very similar to those of acetate kinase gene. The cloned sequence was putatively acetate kinase gene DNA fragment from B49 strain. The results indicated that these degenerate primers de signed by the CODEHOP software could be used to obtain specific gene fragment. Successfully cloning this gene fragment would give the scientific warrant for the metabolic engineering research and reducing the acidification of it's reaction.