Composite

Part:BBa_K2558206:Design

Designed by: Tsinghua 2018   Group: iGEM18_Tsinghua   (2018-10-05)


CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI


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
    Illegal NheI site found at 4626
    Illegal NheI site found at 4649
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5830
    Illegal BamHI site found at 3745
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

After we clarified the correlation between lacI dosage and its induction efficiency, we furthered the experiments by adding CRISPRi to the system. Instead of sfGFP, the IPTG induction devices with different levels of lacI generator is now going to produce a gRNA that targets the promotor of a constitutive sfGFP generator (BBa_K2558206, BBa_K2558207, BBa_K2558208). By adding IPTG, we can induce the transcription of gRNA. Binding with constantly expressed dCas9, gRNA is going to inhibit the expression of sfGFP. We will be able to observe how this process can operate under different levels of lacI expression.


Source

[1] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61.
[2] iGEM parts: PHIF repressible promotor (http://parts.igem.org/Part:BBa_K1725000)

References