CRISPRi Safety Catch device with lux pR promotor
This construct is the “Safety Catch” test system, which is composed of lux pR controlled lacI, constantly expressed dCas9 and IPTG inducible gRNA that specifically binds to lux pR promoter on "Neon" system. Without AHL stimulation, gRNA is produced at a basal level and inhibit luxI and GFP (promoter: lux pR) transcription. Upon the addition of AHL, lacI on Safety Catch is transcribed (here we need to mention that even though the lacI on the Safety Catch is also controlled by lux pR, the design of gRNA allows us to make it unaffected by CRISPRi, thus it can still be activated by the luxR-AHL complex.) and prevents gRNA synthesis, thus relieving inhibition on lux positive feedback system on Neon. We designed this device along with K2558216, which is the same except for the strength of expression and some other parameters.
Usage and Biology
Assisted with the experiences we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid (BBa_K2558214), and Safety Catch the CRISPRi plasmid (BBa_K2558215, BBa_K2558216) work together.
We are still calibrating the NEON system. The results are preliminary, however from Figure.1 we can conclude that the system works to some extent. The positive feedback plasmid Neon (BBa_K2558214) had the highest expression due to uncontrollable leakage. Original lux pR (BBa_R0062) and the new lux pR-HS (BBa_K2558001) we designed had lower basal expression. The addition of Safety Catch (BBa_K2558215) and Safety Catch-HS (BBa_K2558216) almost eliminated the leakage of both positive feedback and the non-positive feedback systems. It is foreseeable that with appropriate parameters NEON system can be activated to almost 10^4 fold.
- Transform the plasmids into E. coli DH5α.
- Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
- Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
- Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
- Each group should be repeated for at least 3 times.
Afroz, T., & Beisel, C. L. (2013). Understanding and exploiting feedback in synthetic biology. Chemical Engineering Science, 103(11), 79-90.
Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., & Arkin, A., et al. (2013). Repurposing crispr as an rna-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1163
- 21Illegal BglII site found at 5547
Illegal BamHI site found at 3442
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 5777
- 1000Illegal BsaI.rc site found at 4314