Overview

This year we improved the part luxR (BBa_C0062). The Vibrio fischeri luminescence genes are activated by an autoinducer and the LuxR protein containing 250-amino acid residue. LuxR is a regulator involved in quorum sensing system. luxR (△2-162) (BBa_K4767000) was constructed by deleting 2-262 amino acids (AHL (acyl homoserine lactone) binding domain) in the N-terminal domain and reserving a C-terminal domain with the function of activating transcription.

Specific process

In the lux system, LuxI produces 3OC6HSL (AHL), which diffuses in and out of the cells. The receptor LuxR and 3OC6HSL form LuxR-3OC6HSL complexes which associate further to polymers. After binding of the polymer to the lux operon, it positively regulates the gene transcription of luxI. As LuxI (in contrast to the LuxR) is encoded on the lux operon, the system contains a positive feedback. The operon constitutively produces the autoinducer in low amounts. If the cell density increases, the positive feedback loop is induced, resulting in an increased autoinducer production.

In order to construct a positive feedback circuit which does not require 3OC6HSL produced by LuxI, we engineered LuxR by deleting 2-262 amino acids in the N-terminal domain (AHL binding domain) and reserving a C-terminal domain with the function of activating transcription, obtaining a resulting regulator luxR (△2-162). LuxR (△2-162) can active the gene transcription driven by the lux promoter in the absence of AHL. To construct the amplifier, we cloned gfp and luxR (Δ2-162) behind the lux promoter. In this design, LuxR (Δ2-162) functions in a positive feedback loop as it can bind to the P_luxI promoter and activate its own transcription.

Fig.1 Positive-feedback loops of modified LuxR (△2-162)

To evidence that the modified LuxR (△2-162) is able to activate gene transcription from the lux promoter without AHL, we cloned gfp at the downstream of the P_luxI -luxR (△2-162). To compare with the original lux system, the gene circuit P_BAD-luxI-P_tac-luxR-P_luxI-gfp, provided by Dr. Qiang Tang from University of Science and Technology of China, was used as the control. In this control gene circuit, the expression of luxI for AHL production and luxR are controlled by arabinose-inducible promoter P_BAD and IPTG-inducible promoter P_tac, respectively. We separately transformed P_luxI-luxR(△2-162)-gfp and P_BAD-luxI-P_tac-luxR-P_luxI-gfp to the S. oneidensis MR-1 which doesn’t contain the lux quorum sensing system.

Fig.2 Modified LuxR(△2-162) can activiate the gfp expression without AHL

As the Figure shows, the cells containing P_BAD-luxI-P_tac-luxR-P_luxI-gfp were able to produce fluorescence only when both IPTG and arabinose were added to the culture. It indicated that IPTG-induced expression of luxR only can active the gfp transcription from P_luxI when arabinose-induced LuxI produces AHL. The LuxR(△2-162) group, however, drive the expression of downstream gfp without any inducers. The results suggest that LuxR(△2-162) can drive downstream gene of the lux promoter constitutively.

Reference

[1] GREENBERG, S.H.C.A., The C-terminal region of the Vibrio fischeri LuxR protein contains. 1991.

[2] Nistala, G.J., et al., A modular positive feedback-based gene amplifier. J Biol Eng, 2010. 4: p. 4.

[3] Hicks M, Bachmann T T, Wang B. Synthetic biology enables programmable cell‐based biosensors[J]. ChemPhysChem, 2020, 21(2): 132-144.

[4] Li F H, Tang Q, Fan Y Y, et al. Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis[J]. Proceedings of the National Academy of Sciences, 2020, 117(37): 23001-23010.