Applying the λ-Red Recombinase System to Generate an rcsF Deletion in Escherichia coli DH300

09/01/2016

Ariel Yi Hsuan Huang, Eva Yi Lei Luan, Nadine Chan, Milo Jinho Yu

Volume 20
Fall 2015 / Winter 2016

The outermembrane sensor protein, RcsF, is a component of the Rcs phosphorelay pathway of Escherichia coli, which regulates capsule and biofilm formation. RcsF is shown to be necessary to sense peptidoglycan disruption in the event of exposure to subinhibitory concentrations of antibiotics. However, the role of RcsF in sensing other forms of environmental stress is unclear. In this study, we apply the λ-Red recombinase system to knockout rcsF in E. coli DH300, a strain that contains rprA, a gene transcribed downstream of the Rcs pathway, fused with a lacZ reporter. We have transformed the λ-Red recombinase-expressing pKD46 into E. coli DH300 using the Transformation and Storage Solution protocol, and PCR was performed to amplify a chloramphenicol resistance cassette that can be used to knock out rcsF through homologous recombination. The expression of rprA after induction with subinhibitory concentration of cefsulodin was measured using the β-galactosidase assay. Our results confirmed that rprA::lacZ fusion in E. coli DH300 is functional. A 1.55 times increase in rprA expression in the induced sample compared to the uninduced control was observed. This study provides preliminary work for future research investigating the role of RcsF.