FRUIT,a Scar-Free System for Targeted Chromosomal Mutagenesis,Epitope Tagging,and Promoter Replacement in Escherichia coli and Salmonella enterica
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| Authors: | Anne M Stringer Navjot Singh Anastasiya Yermakova Brianna L Petrone Jayaleka J Amarasinghe Lucia Reyes-Diaz Nicholas J Mantis Joseph T Wade |
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| Institution: | 1. Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.; 2. Department of Biomedical Sciences, University at Albany, Albany, New York, United States of America.; 3. UASRP CSTEP Program, University at Albany, Albany, New York, United States of America.; University of Birmingham, United Kingdom, |
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| Abstract: | Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of “scar” DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria. |
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