Steps towards construction of a plasmid to express antisense RNA directed at the ribosomal binding site of wbbL in Escherichia coli DFB1655 L9

Abstract

The O antigen oligosaccharide in the outer membrane of Escherichia coli has been found to confer resistance against T4 bacteriophage, thereby preventing cell lysis. O antigen production is regulated by genes found in the rfb gene cluster, including bbl. which encodes for WbbL, a rhamnose transferase. Chow et al. developed an antisense RNA (asRNA) silencing model targeting wbbL to determine if silencing this gene in E. coli K-12 substrain, DFB1655 L9, would increase susceptibility to T4 bacteriophage but did not test functionality. Our project builds upon the work of Chow et al. by adapting and improving the screening process of their asRNA construct. Our approach is to insert the adapted asRNA into an empty pHN678 plasmid vector, transform Escherichia coli TOP10 competent cells with our plasmid, and screen for the presence of our insert through gel electrophoresis. Through screening our colonies for the ligated plasmid on a gel, we found evidence for the presence of our insert. However, attempts at Sanger sequencing of pMOD-wbbL(a) to confirm presence of the insert were inconclusive. Thus, further experiments are needed to test our asRNA silencing model targeting wbbL, with the aim of determining if this affects O antigen production or T4 bacteriophage resistance.