An improved Escherichia coli strain to host gene regulatory networks involving both the AraC and LacI inducible transcription factors

Abstract

Many of the gene regulatory networks used within the field of synthetic biology have extensively employed the AraC and LacI inducible transcription factors. However, there is no Escherichia coli strain that provides a proper background to use both transcription factors simultaneously. We have engineered an improved E. coli strain by knocking out the endogenous lacI from a strain optimal for AraC containing networks, and thoroughly characterized the strain both at molecular and functional levels. We further show that it enables the gradual and independent induction of both AraC and LacI in a simultaneous manner. This construct will be of direct use for various synthetic biology applications.

The mechanisms of gene regulatory networks constrain evolution: A lesson from synthetic circuits

“…Ligation (“blue” and “green” regulatory regions) and Gibson assembly reaction products (“red” regulatory region) were transformed into electrocompetent MK01 cells (Kogenaru and Tans, 2014) that carried already the two other plasmids necessary to complete the synthetic circuit. Transformants were plated out on SM-agar plates.…”

“…The extracted plasmid libraries were mixed with plasmids containing no mutations in a ratio of 70:30 to generate mutant circuits that have mutations in two or three genes. The resulting plasmid mix was transformed into electrocompetent MK01 cells (Kogenaru and Tans, 2014). Circuits that had no mutations or only mutations in one gene were not considered in the analysis.…”

“…For SI Fig. 5b the mutated plasmid coding for the “blue” gene was transformed together with the WT plasmids for the “green” and “red” genes into electrocompetent MK01 cells (Kogenaru and Tans, 2014). For SI Fig.…”

Changes in gene expression shift and switch genetic interactions

“…Microbe strain and growth conditions E.coli BW27783 MK01 strain (kindly provided by the M.Isalan lab), modified to homogenously express arabinose-induced genes (Kogenaru and Tans, 2014) was used to express the mutant library. A single colony of the E.coli BW27783 MK01 strain was picked Luria-Bertani (LB) agar plate, grown overnight at LB liquid medium supplemented with chloramphenicol to 14μg/ml concentration at 37°C.…”

“…We chose the E.coli BW27783 MK01 strain (kindly provided by the Isalan lab) (Kogenaru and Tans, 2014), modified to homogenously express arabinose-induced genes, to express the mutant library. A single chloramphenicol-resistant colony of was picked into 4ml LB medium with 2.8μl of 20mg/ml chloramphenicol and let grow for 3.5 hours at 37°C.…”