Part:BBa_K2558216

CRISPRi Safety Catch device with lux pR-HS promotor

This construct is the “Safety Catch” test system, which is composed of luxpR-HS controlled lacI, constantly expressed dCas9 and IPTG inducible gRNA that specifically binds to lux pR-HS promoter on "Neon" system. Without AHL stimulation, gRNA is produced at a basal level and inhibit luxI and GFP (promoter: lux pR-Hypersensitive, or lux pR-HS) 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-HS, 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 K2558215, which is the same except for the strength of expression and some other parameters.

Usage and Biology

The NEON system, including Neon the positive feedback plasmid (https://parts.igem.org/Part:BBa_K2558214), and Safety Catch the CRISPRi plasmid (https://parts.igem.org/Part:BBa_K2558215, https://parts.igem.org/Part:BBa_K2558216), is expected to establish a positive feedback system with low leakage and high sensitivity.

Results

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-HS has the highest expression due to uncontrollable leakage. Original lux pR and the new lux pR-HS we designed had lower basal expression. The addition of Safety Catch and Safety Catch-HS can almost eliminate any leakage. It is foreseeable that with appropriate parameters NEON system can be activated to 104 fold.

Figure.1. T--Tsinghua-BasalExpressionOf LuxQuorumSensingSystems. The fluorescence intensity is measured by flow cytometer at 488 nm. Neon-HS, Safety Catch, Safety Catch-HS and lux pR, lux pR-HS test plasmids are transferred into E.coli DH5α. The higher the fluorescence is, the more basal expression the system has.

Protocol

1. Transform the plasmids into E. coli DH5α according to the design in Table 2.
2. 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.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. 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. Use flow cytometry to measure fluorescent intensity in 1.5 mg/ml Kanamycin treated medium at one hour intervals.
5. Each group is repeated for at least 3 times.

Reference

[1]Afroz, T., & Beisel, C. L. (2013). Understanding and exploiting feedback in synthetic biology. Chemical Engineering Science, 103(11), 79-90.
[2]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

Assembly Compatibility:

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

  Illegal NheI site found at 310
  Illegal NheI site found at 333
  Illegal NheI site found at 1466
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5850
  Illegal BamHI site found at 3745
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 79
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4617