Construction of a pBAD24 Vector Containing σ32: Restoration of Function in σ32 Knockout Escherichia coli

Many regulatory elements in Escherichia coli that mediate a wide array of responses when subjected to external stress have been progressively discovered and characterized. One of many external stresses that induce a specific 32 response in E. coli is heat shock. This response is largely controlled by σ. Studies have shown that the rpo H knockout mutant E. coli K165 exhibits inhibited growth at 37°C, a normal growth temperature for wild type E. 32 coli. This suggests that σ, the transcription factor encoded by rpo H, is responsible for the downstream activation of various heat shock response genes that allow normal cellular function and growth at elevated temperatures. To 32 study the effects of σ, we constructed a plasmid containing rpo H positively regulated through the arabinose induction system, pMWLD13w. We inserted full length rpo H into a conventional pBAD24 cloning expression vector. Phenotype restoration testing showed that E. coli K165 transformed with pMWLD13w had a comparable growth rate to the wild type E. coli SC122.