Part:BBa_K5136042

LexRO

Biology

LexRO is a synthetic light-switchable repressor, based on a novel LOV light sensor domain, RsLOV. In the darkness, LexRO dimerizes and binds to its cognate operator sequence to repress promoter activity. Upon light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates gene expression.

Usage and design

In the darkness, LexRO dimerizes and binds to the cognate operator sequence to repress the activity of pColE408. Upon blue light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates the expression of ccdB， eventually leading to cell death. We used LexRO, pHybrid 2)-114 version, SD7, ccdA, and ccdB to construct the regulation system and obtained the composite part BBa_K5136231, which was assembled on the expression vector pSB4A5.

Characterization

Facing the threat that the unwanted survival and accumulation of engineered bacteria might happen once they escape to opening environment (1), we designed a light-triggered kill switch for biocontainment of the engineered bacteria. Rather than responding to some chemical inducers, the light-triggered kill switch will be turned to ON state after the engineered bacteria is exposed to the light illumination of specific wavelength. We chose a blue light-inducible optogenetic system, LexRO/pColE408 (2), to control the expression of CcdB toxin, in which an additional expression module of CcdA antitoxin was incorporated as well to neutralize the leaky toxin when the kill switch is in OFF state. Here, we firstly characterized the cytotoxicity of CcdB toxin and the blue light-inducible performance of LexRO/pColE408 system respectively, and then tested the killing effect of the blue light-induced kill switch. Further optimization for improving the killing effect of the switch was also tried primarily.

Figure 1 Characterization of blue light-induced LexRO/pColE408. (A) The gene circuit to characterize blue light-responding performance of LexRO/pColE408 system (BBa_K5136237) on pSB4A5 vector. (B) Agarose gel electrophoresis of the colony PCR products of BBa_K5136237_pSB4A5 in E. coli BL21(DE3). (C) The relative fluorescence units (RFU) of bacterial culture subtracted the background fluorescence of growth media, resulting in the RFU_mCherry. p-value: 0.0029 (**).

We turned to characterize the blue light-responding performance of LexRO/pColE408 optogenetic system used in the kill switch. The photosensor LexRO was controlled by a medium constitutive promoter J23106 and a medium RBS SD7 (3) (BBa_K5136045), while the mCherry fluorescent protein (BBa_J06504) was chosen as the reporter under the control of promoter pColE408 (BBa_K5136044) (Figure 1A), thus generating the composite part BBa_K5136237 on the pSB4A5 vector. BL21(DE3) was used to characterize this optogenetic system, and positive transformants were selected and confirmed by colony PCR (Figure 1B) and sequencing. Characterization was carried out in a self-made blue light (460 nm) illumination device. After cultured for about 17 hours upon blue light illumination (with a relative light intensity of 250) or kept in dark condition, red fluorescence intensity (λe_x = 585 nm, λ_em = 615 nm) and OD600 were measured. The normalized fluorescence intensity of “Light” group showed a significant higher value than that of “Dark” group (about 2 times), indicating that this optogenetic system could be induced by blue light (Figure 1C) indeed.

Reference

1. H. An et al., TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. Molecular Cell 74, 891-908.e810 (2019).
2. Li X, Zhang C, Xu X, Miao J, Yao J, Liu R, Zhao Y, Chen X, Yang Y. A Single-component Light Sensor System Allows Highly Tunable and Direct Activation of Gene Expression in Bacterial Cells. Nucleic Acids Res. 2020 Apr 6;48(6):e33
3. Bernard P, Couturier M. Cell Killing by the F Plasmid CcdB Protein Involves Poisoning of DNA-topoisomerase II complexes. J Mol Biol. 1992 Aug 5;226(3):735-45.
4. Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' Microbial Kill Switches for Bacterial Containment. Nat. Chem. Biol. 2016 Feb;12(2):82-6. 
5. Choi J, Ahn J, Bae J, Koh M. Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts. Molecules. 2022 Oct 11;27(20):6798.
6. Lalwani MA, Kawabe H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.
7. Zhang Y, Xue X, Fang M, Pang G, Xing Y, Zhang X, Li L, Chen Q, Wang Y, Chang J, Zhao P, Wang H. Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy. ACS Appl Mater Interfaces. 2022 Oct 19;14(41):46351-46361.
8. Han C, Zhang X, Pang G, Zhang Y, Pan H, Li L, Cui M, Liu B, Kang R, Xue X, Sun T, Liu J, Chang J, Zhao P, Wang H. Hydrogel Microcapsules Containing Engineered Bacteria for Sustained Production and Release of Protein Drugs. Biomaterials. 2022 Aug;287:121619.