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.