The Effects of Mutations Introduced In Vitro into trbA and trbB on Conjugation in Escherichia coli K12
Abstract
I used an in vitro mutagenisis technique to introduce antibiotic gene inserts into two different F plasmid transfer (tra) operon genes. The transfer functions of these genes, trbA and trbB, had not been previously characterized since the genes were discovered only through protein product and DNA sequence analyses of a cloned 2.7 kb F PstI DNA fragment. I started with small clones and insertionally inactivated the trbA and trbB genes by introducing a gene that encodes antibiotic resistance into a restriction site within their DNA sequence. After confirming the construction of the insert containing plasmids, I transformed each of them into a strain that carries a wild type Flac factor and selected recombinant F factors that carried the antibiotic resistance characteristic of the insertion mutation. These recombinants were evaluated for sensitivity to F-pilus specific phages and donor proficiency. My results suggest that insertion of the tetracyline gene into F trbB may result in loss of F piliation and transfer capacity, and that the trbA gene may not be essential in these functions. However, as non-homologous recombination between F and the pKI plasmids could have occurred to give unexpected configurations of F DNA associated with antibiotic resistance, additional experiments are needed to confirm that the antibiotic resistant F factors I identified do indeed contain trbA and trbB mutations.
Description
Program year: 1996/1997Digitized from print original stored in HDR
Citation
Rebolledo, Michael A. (1987). The Effects of Mutations Introduced In Vitro into trbA and trbB on Conjugation in Escherichia coli K12. University Undergraduate Fellow. Available electronically from https : / /hdl .handle .net /1969 .1 /CAPSTONE -RebolledoM _1987.