![]() Gene 219(1–2):91–99ĭatsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Gene 148(1):71–74Ĭhaffin DO, Rubens CE (1998) Blue/white screening of recombinant plasmids in gram-positive bacteria by interruption of alkaline phosphatase gene ( phoZ) expression. doi: 10.1186/1475-īernard P, Gabant P, Bahassi EM, Couturier M (1994) Positive-selection vectors using the F plasmid ccdB killer gene. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.īanerjee S, Kumar J, Apte-Deshpande A, Padmanabhan S (2010) A novel prokaryotic vector for identification and selection of recombinants: direct use of the vector for expression studies in E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. The new blue/white screening system contains a recipient E. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli.
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