Difference between revisions of "Part:BBa K5136235"

Revision as of 09:55, 2 October 2024

J23106-SD17-LexRO-B0015-pColE408-ccdB-B0014-pHybrid 2)-114 version-ccdA

Biology

LexRO

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.

SD17

SD17 is a ribosome binding site on the genome , which is capable of recruiting ribosomes in engineered E. coli.

pHybrid 2)-114

pHybrid 2)-114 version is an engineering promoter that is suppressed by the Aca2 repressor, which uses the -35 and -10 regions of J23114.

pColE408-RBS

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.

ccdB

ccdB gene is located on the Escherichia coli F-factor plasmid and is part of the toxin-antitoxin system encoded by the ccd operon, responsible for plasmid maintenance during cell division. ccdB encodes a toxic protein that acts as a DNA gyrase poison. It can bind DNA gyrase to the broken double-stranded DNA, leading to cell death.

ccdA

ccdA is the gene found within the ccd operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB. CcdA protein is easily degraded by Lonprotease.The cell loses the ccdA gene due to the loss of the F plasmid, causing the cell to succumb to the toxicity of CcdB.

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.

After verifying the cytotoxicity of CcdB and blue light-inducible performance of LexRO/pColE408 system, we built the blue light-induced kill switch (BBa_K5136231), in which the toxin-antitoxin module is controlled by promoter pColE408 and LexRO is constitutively expressed as in BBa_K5136237 (Figure 1A). While the LexRO expression module only (BBa_K5136234) on the pSB4A5 was set as the control. Positive transformants were selected and confirmed by colony PCR (Figure 1B) and sequencing after transformed to BL21(DE3). Spot assay was also performed after cultured upon blue light illumination or kept in dark condition. A blue light illumination-dependent killing effect was observed, which indicates that this blue light-induced kill switch functioned to kill engineered bacteria when exposed to blue light (Figure 1C). Besides, when exposed to blue light for whole period (6 hours, “L”), the kill switch exhibited a slightly stronger killing effect than exposed to blue light for a shorter time (kept in dark for 2 hours 25 min first then switched on the blue light for 3 hours 35 min, “D/L”), which implied that the killing of engineered bacteria might be illuminating time-dependent.

Although we have verified the blue light-dependent killing effect of the kill switch, we still tried to optimize the gene circuit for further improving the killing effect. Since lower LexRO content were more sensitive to light illumination (3), we then changed the RBS of LexRO in the gene circuit to a weaker one (SD17, BBa_K5136049) to see whether this would improve the effect of killing or not, resulting in the generation of BBa_K5136235 composite part on pSB4A5 vector (Figure 2A). Colony PCR (Figure 2B) and sequencing were performed again to confirm the positive transformants of BL21(DE3). Similar test was done to the alternative kill switch. When lower the expression of LexRO, a slight decrease on survival ratio was obtained for the kill switch (Figure 2C), indicating that the strategy for optimizing the kill switch might be available and feasible.

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).

Sequence and Features