Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop

Abstract

The CusSR two-component system (TCS) is a copper-sensing apparatus of E. coli that is responsible for regulating the copper-related homeostatic system. The dynamic characteristics of the CusSR network were modified by the introduction of a positive feedback loop. To construct the feedback loop, the CusR, which is activated by the cusC promoter, was cloned downstream of the cusC promoter and reporter protein. The feedback loop system, once activated by environmental copper, triggers the activation of the cusC promoter, which results in the amplification of a reporter protein and CusR expression. The threshold copper concentration for the activation of the modified CusSR TCS network was lowered from 2,476.5 μg/l to 247.7 μg/l, which indicates a tenfold increase in sensitivity. The intensity of the output signal was increased twofold, and was maintained for 16 h. The strategy proposed in this study can also be applied to modify the dynamic characteristics of other TCSs.

A copper‐specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli

“…Coding sequences of genes were obtained from NCBI, IGEM, and BioCyc databases. gfp and P t7 from our laboratory storage, terminator (BBa_B0014) from 2017 DNA Distribution Kit Plates, OprF from P. aeruginosa PAO1, and ribB and P cusC (Ravikumar, Pham, et al, 2012) from E. coli DH5α were amplified using the primers listed in Table S1 and were then cloned into pLZ01. Subsequently, ribB (or gfp ) was inserted downstream of P cusC , while OprF was fused to P t7 .…”

Synthetic bacteria for the detection and bioremediation of heavy metals

“…The LOD of the system was as low as 12 µM of Cu 2+ . Ravikumar et al developed a positive feedback loop by co-expressing cusR with the gfp gene via P cusC promoter to enhance Cu sensitivity ( Ravikumar et al, 2012 ). The LOD of the biosensor was improved by 10-fold (4 μM) compared with the native system.…”

Heavy Metal Detection and Removal in Artificial Wastewater Using Two-Component System Based Recombinant Bacteria

“…Zn 2+ 와 Cu 2+ 인지를 위한 박테리아 바이오센서는 pZGFP1과 pCGFP1 플라 스미드를 도입한 재조합 대장균을 사용하였다 (Ravikumar et al, 2011a(Ravikumar et al, , 2012. Zn 2+ 와 Cu 2+ 의 제거가 가능한 박테리아로는 pZZ1056과 pCC1056 플라스미드를 도입한 재조합 대장균을 사 용하였다 (Ravikumar et al, 2011a(Ravikumar et al, , 2011b.…”

“…또한 CusS/R TCS는 Cu 2+ 를 감지하여 구리 efflux와 연관된 cusC 유전자를 발현시킨다 (Munson et al, 2000;Leonhartsberger et al, 2001). (Ravikumar et al, 2011a(Ravikumar et al, , 2012. 이때 TCS 기 반 아연 바이오센서는 0.001 mM의 Zn …”