Developing an Antisense RNA Vector for Skp Chaperone Knockdown Analysis in Escherichia coli BW25113

Abstract

Seventeen-Kilodalton Protein (Skp) is a highly conserved periplasmic chaperone protein among Gram-negative bacteria. It plays a crucial role in the folding and assembly of outer membrane proteins (OMPs) in the periplasms. Studies utilizing skp knockout (Δskp) mutant cells found that Skp also likely prevents periplasmic protein aggregation. This study aimed to develop a method to silence skp expression and characterize skp knockdown Escherichia coli cells. We employed anti-sense RNA (asRNA) silencing by constructing a skp asRNA insert and ligating it into a pHN678 vector, which contains an isopropyl-beta-thiogalactopyranoside (IPTG)-inducible promotor. The asRNA insert was designed to hybridize to a 40 base-pair (bp) region of the skp messenger RNA (mRNA) containing the Shine-Dalgarno (SD) ribosome binding site, as well as the first four codons of the coding sequence. Sequencing result of the asRNA vector, pJAHS, confirmed the successful and correct ligation of the insert. To characterize the knockdown cells, pJAHS was transformed into E. coli BW25113 cells for comparisons with Δskp E. coli from the Keio collection. A zone of inhibition functional assay was used to assess the degree of skp knockdown in transformants. The results indicated that the Skp protein levels in pJAHS transformants were somewhere between wild-type (WT) and knockout levels. Growth of cultures at different temperatures suggested that the asRNA hybridization efficiency is greater at cooler temperatures, but this may have been an artifact of poor growth conditions. Lastly, growth-curve analysis of pJAHS transformants at varying concentrations of IPTG resulted in no significant phenotypical differences when compared to WT cells. This model allows for fine control of Skp expression and can guide and be utilized in future research of bacterial chaperone systems.