Part:BBa_K3447133
light-on induced system
In this part, we added a reporter gene sfGFP, thereby characterizing its function by turning off the expression of the sfGFP gene when blue light irradiation.
Improved By Team Songshan-Lake
Group: Songshan-Lake iGEM 2024
New Improved Part:
BBa_K5348004 (pL-RBS1), BBa_K5348005 (pL-RBS2), and BBa_K5348006 (pL-RBS3)
Existing Part:
BBa_K3447133 (Light-on induced system)
Summary:
To construct engineered strains based on light-controlled regulatory systems, we used a light-inducible system (BBa_K3447133, hereafter referred to as pL). However, in the course of our research, we found that there was a leakage of the pL light-control system, which led to the failure construction of the plasmids containing toxin proteins. After literature research and analyzing the results of the pre-experiment, we adopted the RBS replacement strategy to reduce the intensity of the RBS linked to the target gene in the pL element. We verified the effectiveness of this strategy in fluorescent protein mCherry and toxic protein mazF. The results showed that we successfully verified the availability and tunability of the light-control system, and also obtained the optimal combination of pL mutant and toxicity protein mazF, which can be used for the construction of light-controlled algicidal bacteria. These optimized components can also provide theoretical and experimental support for other teams conducting related research.
1. Usage and Biology
The pL light-control system consists of several basic parts. Under the dark condition, the cI gene can be expressed, inhibiting the transcription of PR and the downstream gene cannot be expressed. Under blue light, the cI gene cannot be expressed, PR can be transcribed normally, and the downstream gene can be expressed.
The length and base composition of the RBS sequence affects ribosome binding and translation efficiency to some extent [1]. A stronger RBS makes it easier for ribosomes to bind, thereby increasing gene expression levels, while a weaker RBS can reduce gene expression levels or only express under specific conditions. Therefore, by replacing the RBS sequence in the pL element that regulates the translation of the target protein, we can control the expression level of the target protein (Figure 1). We obtained three mutants of the original RBS (BBa_B0034) in the pL light-control element from the literature [2]. The RBS calculator showed that the strength of these three RBS decreased by 9-fold, 108-fold, and 150-fold, respectively.
2. Characterization/Measurement
(1) pL-RBS(n)-mCherry
a. Construction Design
The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript company, which was divided into two fragments for synthesis, pL-1 and pL-2. Then, we used primers to bring different RBS mutants into the pL element, amplifying pL-2-RBS(n) and RBS(n)-mCherry fragments, respectively, and then connecting these two fragments through overlap PCR to obtain the pL-2-RBS(n)-mCherry fragments (Figure 2B). The agarose gel electrophoresis results showed that we successfully obtained the above fragments. Then we connected pL-1, pL-2-RBS(n)-mCherry fragments, and the pTrc99k vector through Gibson assembly. Colony PCR and sequencing results confirmed that we constructed these four plasmids: pYC-pKC-pL-RBS(0/1/2/3)-mCherry (Figure 2C-F).
b. Measurement: Light Control Test
Subsequently, we conducted light-control tests on four strains containing pYC-pKC-pL-RBS(0/1/2/3)-mCherry plasmids, respectively. We cultured the strains under dark condition and blue light irradiation, respectively, sampling at intervals to measure the RFU (relative fluorescence units) of the bacterial suspension. As shown in Figure 3, the test results verified that the pL light-control element could regulate mCherry expression under dark and blue light conditions. As the RBS strength decreased, the RFU of mCherry decreased accordingly, indicating that the RBS replacement strategy can achieve regulation of the pL light-control system (Figure 3E).
However, it was observed that the pL light-control system exhibited leakage, with detectable increases in mCherry RFU after culturing for more than 8 hours under dark conditions. It was also noted that reducing the RBS strength mitigated the leakage situation of the pL light-control system (Figure 3B-C). No fluorescence values were detected for pL-RBS(3)-mCherry when cultured in both dark and blue light conditions, indicating that the strength of this RBS was too low (decreased by 150-fold) to initiate mCherry translation (Figure 3D).
(2) pL-RBS(n)-MazF
a. Construction Design
To achieve light-controlled regulation of bacterial growth, we replaced the target protein following the pL element with the toxic proteins MazF, which can inhibit protein synthesis by cleaving mRNA [3]. To obtain these plasmids, we amplified the pL-1, pL-2-RBS(n), RBS(n)-MazF, and pTrc99k backbone fragments, respectively. To improve the efficiency of homologous recombination, we first used overlap PCR to obtain the pL-2-RBS(n)-MazF fragments, and then we homologous recombined the pL-1, pL-2-RBS(n)-MazF, and pTrc99k backbone fragments (Figure 4A). The agarose gel electrophoresis results showed that we successfully obtained pL-2-RBS(n)-MazF fragments through overlap PCR (Figure 4B).
For the pYC-pKC-pL-RBS(n)-MazF plasmids, we transformed it into E. coli DH5α competent cells. Colony PCR and sequencing results confirmed the successful construction of pYC-pKC-pL-RBS(2/3)-MazF plasmids (Figure 5). However, plasmids containing original RBS (RBS0) and RBS1 failed to construct, with colony PCR revealing fragment deletions in the target pL-RBS(0/1)-MazF segments (Figure5A).
b. Measurement: Light Control Test
We conducted light-control tests on the successfully constructed strains. Results showed that under blue light cultivation, pL-RBS(2/3)-MazF reduced bacterial concentration (OD600) by 1.6 and 1.2 times, respectively, compared to dark conditions. This indicates that under blue light, the toxic protein MazF was successfully expressed and inhibited bacterial growth, demonstrating that the pL element can regulate MazF expression. The group containing RBS2 showed more pronounced inhibition than RBS3, due to the difference in RBS strength (Figure 6).
3. Learn
In conclusion, we verified that the pL light-control system can successfully regulate mCherry expression in E. coli DH5α, and the RBS replacement strategy can effectively modulate the strength of this light-control system. This provides an experimental foundation for the construction of light-controlled algicidal bacteria. Furthermore, we conclude that the combination of RBS2 and the toxic protein MazF is most suitable for constructing light-controlled algicidal bacteria.
We also analyzed the reason for the failure of the pL-RBS(0/1)-MazF construct, presumably due to leakage of the pL light-control element, leading to leaky expression of MazF. Due to the high toxicity of MazF, leaky expression occurs even when the RBS strength is reduced by 9-fold (RBS1). This causes survival pressure on the bacteria, which results in no correct transformants.
Reference
[1] Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene, 1999, 234(2), 187-208.
[2] Ji W, Shi H, Zhang H, Sun R, Xi J, Wen D, Feng J, Chen Y, Qin X, Ma Y, Luo W, Deng L, Lin H, Yu R, Ouyang Q. A formalized design process for bacterial consortia that perform logic computing. PLoS One. 2013;8(2): e57482.
[3] Christensen SK, Pedersen K, Hansen FG, Gerdes K. Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. Journal of Molecular Biology. 2003;332(4):809–819.
===Usage and Biology=== YF1 is the kinase for FixJ in the blue light system. Without blue light irradiation, YF1 phosphorylates FixJ, activating the downstream expression after promoter PFixK2. Once the blue light is on, the FixJ cannot be phosphorylated, shutting down the downstream gene expression.===Characterization=== [[Image: Pathway of Blue Light-on System.jpg|thumb|center|500px|Fig. 1 Pathway of Blue Light-on System. (A) without blue light; (B) with blue light.]]
To enrich the blue-light sensing system, repressor pair CI-Plambda were introduced in our project to achieve the goal of blue light-on switch system (click to [https://2020.igem.org/Team:Jilin_China/Improvement Jilin_China 2020 Improvement]). Therefore, in our blue light-on system, when blue light is on, PFixK2 is blocked and Plambda can turn on the expression of sfGFP (Fig. 1A). Without blue light, CI functions normally thereby inhibiting the expression of downstream sfGFP (Fig. 1B).
Based on the experiments above, to verify the function of blue light-on system, two control groups with mock were set to proof the activation of the blue light on our bacteria. As is shown in Fig. 2, compared with the construct without blue light, our blue light-off system would constantly activate the fluorescent expression with blue light induced. [[Image: Blue Light-on system can regulate the downstream gene via switch of light.jpg|thumb|center|500px|Fig. 2 Blue Light-on system can regulate the downstream gene via switch of light. (A) The emission intensity at 528 nm was measured at the excitation wavelength of 485 nm. After that, measure these values at the indicated time. (B) E. coli DH5α with blue light-on system. (a) 18-hour incubation without blue light; (b) 18-hour incubation with blue light.]]
==Design== ===Design Notes=== We added some synonymous mutations to avoid part rules.
===Source=== We found this sequence data in GenBank.
===References=== Sequence and Features
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