Construction of a T-Vector Using an Esterase Reporter for Direct Cloning of PCR Products

• Published in: Journal of microbiology and biotechnology Vol. 20; no. 11; pp. 1481 - 1483
• Main Authors: Lim, H.D., Chonnam National University, Gwangju, Republic of Korea, Cheong, D.E., Chonnam National University, Gwangju, Republic of Korea, Shin, H.J., Chosun University, Gwangju, Republic of Korea, Kim, G.J., Chonnam National University, Gwangju, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.11.2010; 한국미생물·생명공학회
• Subjects: Base Sequence; Biological and medical sciences; Biotechnology; Cloning, Molecular - methods; ESTERASAS; ESTERASE; ESTERASES; Esterases - genetics; Esterases - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Genetic Vectors - genetics; Genetic Vectors - metabolism; Molecular Sequence Data; Mutagenesis, Insertional; PCR product; Polymerase Chain Reaction; Protein Engineering; reporter; T-vector; TA cloning; 생물학; Polymerase chain reaction; PCR product; Esterase; TA cloning; Enzyme; Hydrolases; T-vector; Esterases; reporter; Vector
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1007.07015

We constructed an efficient T-vector, pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones owing to insertional inactivation of the esterase reporter. Additionally, PCR products were efficiently cloned into this vector without the gel purification steps, owing to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.