An investigation into the inability of lactose to induce the pET expression system in a sub-strain of Escherichia coli BLR(DE3).

摘要

In genetically modified Eschericia coli (E. coli) the lactose (lac) promoter is commonly used to regulate the expression of foreign genes. In laboratory cultures a lactose analog isopropyl B-D-thiogalactopyranoside (IPTG) is routinely used to induce these genes. However, for economical reasons lactose is commonly used in larger fed-batch cultures where its concentration can be maintained. An efficient method of producing recombinant proteins in E. coli is the pET (plasmid for expression by T7 RNA polymerase) system. BL21 is a B strain of E. coli, which is a widely used host for this expression system and T7 RNA polymerase expression in BL21(lambdaDE3) is inducible with either IPTG or lactose. BLR is a recA deletion mutant of BL21 however T7 RNA polymerase expression in a sub-strain of BLR(lambdaDE3) is inducible only with IPTG. To investigate the inability of lactose to induce the pET system in this sub-strain (test strain), experiments were carried-out comparing it to isogenetic strains (control strains).; Using western bolt analysis, blockage in the expression cascade was determined to be at the induction of T7 polymerase expression. While in the controls, there was no LacZ activity detected in the test strain even in the absence of glucose and the presence of both IPTG and cAMP. Based on these observations the integration site of the DE3 prophage was confirmed by PCR. Catabolite repression was ruled-out as a reason for the absence of LacZ activity by cultivating strains on different carbon and energy sources. While the controls were able to grow on minimal media, the test strain required isoleucine (ilvA -) and was unable to grow on lactose or culture media with low potassium levels. Finally BLR was lysogenized with the DE3 phage and resulting BLR(lambdaDE3) lysogens were able to grow on minimal media containing lactose as the sole carbon and energy source. Based on these findings it can be concluded that the inability of lactose to induce the pET system in the original BLR(lambdaDE3) strain was not the direct result of Delta recA but was more likely due to a random spontaneous mutation in lacZ.