Developing the Antisense Silencing Model for the Investigation of the Mechanism of Resistance of Escherichia coli DFB1655 L9 to T4 Bacteriophage


Derek Chow, Amanda Clark, Yuan Hung (Oliver) Huang, Si Zhe (Celeste) Ng

Volume 24
Fall 2018 / Winter 2019

O antigen consists of multiple repeating polysaccharide units found on lipopolysaccharide in some Gram-negative bacteria, including Escherichia coli. It is
considered to be a virulence factor as well as a point of interaction with certain bacteriophages. The E. coli substrain MG1655 has an insertion of an IS5 element within wbbL, preventing WbbL expression, thus interrupting the O antigen synthesis pathway. DFB1655 L9 is generated from the isogenic strain MG1655 to restore functional wbbL via a single crossover homologous recombination. MG1655 is susceptible to T4 bacteriophage infection whereas DFB1655 L9 is resistant, however the mechanism of DFB1655 L9 resistance is unknown. In this study, we hypothesize that the insertion of wbbL and the subsequent expression of O16 antigen in DFB1655 L9 confer resistance to T4 infection. We confirmed that DFB1655 L9 is resistant to T4 infection up to a multiplicity of infection of
20. We also designed pCODA-wbbL(a) for antisense RNA silencing of wbbL by cloning the antisense ribosome binding site of wbbL into pHN678 and transforming into DFB1655 L9.