Development of a novel T-vector supporting efficient green-white screening

Abstract

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector.