Difference between revisions of "Part:BBa K3447112"

Revision as of 10:09, 27 October 2020

light-off induced system

This part can turn off the expression of the sfGFP gene when blue light irradiation.

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

In the blue light-off system, YF1 switched to turn on in the dark and phosphorylate FixJ, which binds to the P_FixK2 to initiate expression of the downstream sfGFP. When irradiated with blue light, the phosphorylation of YF1 was repressed, thereby inhibiting the downstream sfGFP (Fig. 1A & B).

Fig. 1 Digestion and electrophoresis of YF1-fixJ-P_FixK2-sfGFP.

To verify the construction and function of blue light-off system, two groups were set to proof the strong inhibition of the blue light on our bacteria. Moreover, compared with the construct without blue light, our blue light-off system would constantly inhibit the fluorescent expression with blue light induced. And since the construct shows little difference in fluorescent expression with the mock when blue light on, we indicated our system with a low leak (Fig. 2 and 3).

Fig. 2 Digestion and electrophoresis of YF1-fixJ-P_FixK2-sfGFP.

Fig. 3 Blue Light-off system can regulate the downstream gene via switch of light. (A) The fluorescence detection of blue light-off system. E. coli DH5α was transformed with the designed plasmid, cultured for 8 hrs, and diluted OD₆₀₀ to 0.04. Treatment with or without blue light (λ= 460 nm) irradiated respectively for detection of the fluorescence intensity of sfGFP at the indicated time. (B) E. coli DH5α with blue light-off system. (a)12-hour incubation without blue light; (b) 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