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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1882
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 587
  Illegal NgoMIV site found at 659
  Illegal NgoMIV site found at 749
  Illegal NgoMIV site found at 767
  Illegal NgoMIV site found at 1259
  Illegal NgoMIV site found at 1552
  Illegal NgoMIV site found at 1646
  Illegal AgeI site found at 301
  Illegal AgeI site found at 1427
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1316
  Illegal BsaI.rc site found at 200
  

Source

We found this sequence data in GenBank.

Design

Design Notes

We added some synonymous mutations to avoid part rules.

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 P_FixK2. Once the blue light is on, the FixJ cannot be phosphorylated, shutting down the downstream gene expression.

Characterization

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-P_lambda were introduced in our project to achieve the goal of blue light-on switch system (click to Jilin_China 2020 Improvement). Therefore, in our blue light-on system, when blue light is on, P_FixK2 is blocked and P_lambda 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.

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.

References

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.

Contribution By Team Songshan-Lake

Summary

We have added new experimental data to the already existing part: BBa_K3447133 (light-on induced system). We used mCherry as a reporter gene and therefore added a new composite part: BBa_K5348008, to test the light control system.

Construction Design

This composite part consists of the BBa_K3447133 (hereafter referred to as the pL-RBS0) and fluorescent protein mCherry (BBa_K3822002). With the pL light-control system, regulation of mCherry expression in the dark and under blue light can be achieved.

Engineering Principle

The pL light-control system consists of several basic parts. Under dark condition, histidine kinase (YF1) phosphorylates FixJ (response regulator of histidine kinase), which activates P_FixK2 (the target gene for transcription upon FixJ activation), driving the expression of cI gene (λ phage repressor), which represses the transcription of its cognate promoter, P_R (the cognate promoter of cI), and downstream genes cannot be expressed. Under blue light, the cI gene cannot be expressed, P_R can be transcribed normally, and downstream genes can be expressed [1].

Figure 1. Schematic diagram of pL-RBS0-mCherry.

Experimental Approach

The plasmid construction scheme is shown in Figure 2A. We synthesized the pL element at GenScript and divided it into two fragments, pL-1 and pL-2, for synthesis. We amplified pL-1, pL-2-RBS(0), and RBS(0)-mCherry fragments, and then ligated the pL-2-RBS(0) and RBS(0)-mCherry fragments by overlapping PCR to obtain the pL-2-RBS(0)-mCherry fragment. Finally, we ligated pL-1, pL-2-RBS(0)-mCherry fragments, and pTrc99k vector by Gibson assembly. Colony PCR and sequencing results confirmed that we constructed the pYC-pKC-pL-RBS(0)-mCherry plasmid (Figure 2B).

Figure 2. Construction results of pYC-pKC-pL-RBS(0)-mCherry plasmid. (A) Construction Strategy. (B) Colony PCR and sequencing results.

Measurement: Light Control Test

Subsequently, we conducted light-control tests on the strain containing pYC-pKC-pL-RBS(0)-mCherry plasmid. We cultured the strains under dark conditions 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. 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.

Figure 3. Light-control test results.

References

[1] 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.

Improved By Team Songshan-Lake

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-RBS0). However, in the course of our research, we found that there was a leakage of this light-control system, which led to the failure in constructing plasmids containing toxin proteins. Thus, we adopted the RBS replacement strategy to reduce the intensity of the RBS linked to the target gene in the pL element.

We added new parts: BBa_K5348004 (pL-RBS1), BBa_K5348005 (pL-RBS2), and BBa_K5348006 (pL-RBS3), and tested them using mCherry. The results showed that we could successfully modulate the intensity of the light control system (Figure 1). Furthermore, we found that the combination of the BBa_K5348005 part (pL-RBS2) and MazF is the most suitable for constructing light-controlled bacteria (Figure 2).

Figure 1. Light-control tests of pL-RBS(n)-mCherry plasmids.

Figure 2. Light-control tests of pL-RBS(2)-MazF.

References

[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.