title: Construction of a directional T vector for cloning PCR products and expression in Escherichia coli.
abstract: In order to clone PCR products and express them effectively in Escherichia coli, a directional cloning system was constructed by generating a T vector based on pQE-30Xa. The vector was prepared by inserting an XcmI cassette containing an endonuclease XcmI site, a kanamycin selective marker, a multiple-cloning-site (MCS) region and an opposite endonuclease XcmI site into the vector pQE-30Xa. The T vector pQE-T with single overhanging dT residues at both 3' ends was obtained by digesting with the restriction enzyme XcmI. For directional cloning, a BamHI site was introduced to the ends of the PCR products. A BamHI site was also located on the multiple cloning site of pQE-T. The PCR products were ligated with pQE-T. The directionally inserted recombinants were distinguished by using BamHI to digest the recombinants because there are two BamHI sites located on the both sides of PCR fragment. In order to identify the T-vector functions, the 14-3-3-ZsGreen and hRBP genes were amplified and a BamHI site was added to the ends of the genes to confirm this vector by ligation with pQE-T. Results showed that the 14-3-3-ZsGreen and hRBP were cloned to the vector pQE-T directly and corresponding proteins were successfully produced. It was here demonstrated that this directional vector is capable of gene cloning and is used to manipulate gene expression very easily. The methodology proposed here involves easy incorporation of the construct into other vectors in various hosts.
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
authors: Xiu-Yi
authors: XY
authors: Liang
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
authors: Zhi-Cheng
authors: ZC
authors: Liang
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
authors: Zhi
authors: Z
authors: Zhang
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
authors: Jiao-Jiao
authors: JJ
authors: Zhou
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China.
authors: Shi-Yu
authors: SY
authors: Liu
authors: Shenzhen Key Laboratory of Microbial Genetic Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, China. Electronic address: sltian@szu.edu.cn.
authors: Sheng-Li
authors: SL
authors: Tian
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D007612:Kanamycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D011994:Recombinant Proteins; D007612:Kanamycin; C084267:endodeoxyribonuclease XcmI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Directional cloning; Prokaryotic expression; T vector; XcmI cassette