Accurate insertional inactivation of lacZalpha: construction of pTrueBlue and M13TrueBlue cloning vectors

Gene
S N Slilaty, S Lebel

Abstract

Color selection plasmid and phage vectors based on insertional inactivation of the lacZalpha gene fragment have been in wide use for nearly two decades. The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZalpha placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZalpha-complementation peptide. Numerous observations found in the literature and made in our laboratory suggest that the color selection process in these vectors does not function reliably and that erroneous conclusions about the success of cloning experiments are frequently drawn. We have studied the efficiency of lacZalpha inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene (lacZalpha). Our results show that accurate and reliable inactivation of lacZalpha occurs only when insertions are made within the DNA region that encodes aa 11-36. Based on these results, novel color selection plasmid and phage vectors, pTrueBlue and M13TrueBlue, have been constructed. Application of thes...Continue Reading

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Citations

Oct 16, 2002·Biochemical and Biophysical Research Communications·Nadezhda GutkinaSergei P Kovalenko
Feb 12, 2008·Journal of Bacteriology·Diederik Johannes OppermanEsta van Heerden
Jul 28, 2004·Proceedings of the National Academy of Sciences of the United States of America·Ido Golding, Edward C Cox
Mar 20, 2012·Analytical Biochemistry·Dea-Eun CheongGeun-Joong Kim
Apr 18, 2013·Protein Science : a Publication of the Protein Society·Elizabeth B Speltz, Lynne Regan
Feb 24, 2015·Protein Expression and Purification·Xinxin LiuPeng Liang
Jun 3, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Margarita Daniela Tafoya-RamírezBernardo Franco

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