Regulatory

Part:BBa_K3249000

Designed by: Omar El-Fahmawi   Group: iGEM19_UC_Davis   (2019-10-04)


CIBN-dCas9-CIBN

This part is composed of a deactivated Cas9 protein (dCas9) flanked by two CIBN regions intended for use in the light activated CRISPR/Cas9 effector system (LACE). When induced by blue light, CRY2 dimerizes with its binding partner CIBN, effectively bringing the transcriptional activator VPR to the target site defined by the dCas9/gRNA complex. gRNAs can be designed in order for the dCas9 protein to target and increase transcription of specific genes. A figure of how the system works is shown below.

Figure 1:
Light activation triggers the dimerization of CRY2 to CIBN, which brings the activator VP64 to the promoter region of the GOI increasing rate of transcription.

Characterization

We used lipofectamine to transiently cotransfect CHO cells with 2 plasmids. One containing CIBN-dCas9-CIBN and our 3 gRNAs and the other containing CRY2-VP64. When both plasmids are successfully transfected into the cells we expect to see an increase in transcription for the gene IL1RN when the cells are induced by blue light. The cells were incubated at 37° C for 24 hours to allow them to adhere to the 24 well plate. After 24 hours, they were placed into our light plate apparatus (LPA) while still being incubated at 37°C. The LPA illuminated the wells with blue light for 24 hours. We then extracted the RNA from the CHO cells and used reverse transcriptase to convert this RNA into cDNA. Using the cDNA as template DNA, qPCR was performed to quantify the fold increase of IL1RN expression compared to untreated cells.

Figure 1:
Fold expression of IL1RN in CHO cells exposed to different light intensities.

Fold increase was calculated using the delta-delta Ct method for qPCR. All data was normalized to the reference genes recommended by MIQE guidelines and fold increase was compared to untransfected cells. A more in depth description of experimental conditions and data analysis can be found on the UC Davis 2019 iGEM wiki.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1360
    Illegal PstI site found at 2782
    Illegal PstI site found at 2986
    Illegal PstI site found at 3016
    Illegal PstI site found at 4228
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4717
    Illegal PstI site found at 1360
    Illegal PstI site found at 2782
    Illegal PstI site found at 2986
    Illegal PstI site found at 3016
    Illegal PstI site found at 4228
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 821
    Illegal BamHI site found at 4708
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1360
    Illegal PstI site found at 2782
    Illegal PstI site found at 2986
    Illegal PstI site found at 3016
    Illegal PstI site found at 4228
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1360
    Illegal PstI site found at 2782
    Illegal PstI site found at 2986
    Illegal PstI site found at 3016
    Illegal PstI site found at 4228
    Illegal NgoMIV site found at 109
    Illegal NgoMIV site found at 1648
    Illegal NgoMIV site found at 2752
    Illegal NgoMIV site found at 2825
    Illegal NgoMIV site found at 3310
    Illegal NgoMIV site found at 4219
    Illegal NgoMIV site found at 4867
  • 1000
    COMPATIBLE WITH RFC[1000]


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