(ii) Construction of new vectors
pWY51 (pCT1). A 821-bp fragment containing the tetR gene encoding the TetR repressor and the tetR promoter region (tetR+ -Ptet ) was PCR-amplified from pYJ335 (Ji et al.,1999) using primers oWY466 and oWY467. pCL55 (Lee et al., 1991) was used as the backbone plasmid and was amplified by PCR using primers oWY468 and oWY469. The DNA fragments were assembled by the Gibson method (Gibson et al., 2009), inserting thetetR+ -Ptet cassette at the multicloning site of pCL55, and the DNA mixture was transformed into E. coli DH5α.
pWY52 (pCT2), and pWY53 (pCT3). A 2225-bp fragment containing thetetM. gene from pTET (Bose et al., 2013) was amplified by PCR using primers oWY486 and oWY487. pCL55 or pWY51 as a backbone plasmid was amplified by PCR using primers oWY488 and oWY489 .The DNA fragments were assembled by the Gibson method and the DNA mixture was transformed into E. coli DH5α, resulting in pWY52 (pCT2) or pWY53 (pCT3) that confer tetracycline resistance instead of chloramphenicol resistance.