Part:BBa_K5136044

pColE408-RBS

Biology

pColE408-RBS is a combination of the promoter pColE408 and RBS. PcolE408 is a promoter to which LexRO (BBa_K5136042) binds and represses the expression of genes driven by the promoter. The promoter mainly consists of two parts: ColE promoter, which is a strong promoter, and LexA(408)-operator, which is the binding site of LexRO.

Usage

pColE408-RBS was used to initiate the expression of ccdB, CcdB locks DNA gyrase together with broken double-stranded DNA, ultimately leading to cell death. At the same time, we used pColE408-RBS to initiate the expression of aca2 and inhibit the expression of the underlying CcdA protein. 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. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018). 2. H. Owji, N. Nezafat, M. Negahdaripour, A. Hajiebrahimi, Y. Ghasemi, A Comprehensive Review of Signal Peptides: Structure, Roles, and Applications. Eur. J. Cell Biol. 97, 422–441 (2018). 3. L. A. Fernández, I. Sola, L. Enjuanes, V. De Lorenzo, Specific Secretion of Active Single-chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System. Appl. Environ. Microbiol. 66, 5024–5029 (2000).