Part:BBa_K3249007
CHO IL1RN sgRNA 1-3
Usage and Biology
This part consists of 3 single guide RNAs cloned into the gRNA scaffold of addgenes pX330A1x3 plasmid. Downstream of the gRNA scaffold is a U6 promoter, which is constitutively active. We used a total of 3 sgRNAs in our CRISPR/dCas9 system in order to increase the likelihood of gRNAs binding to the promoter site.
We tested this combination of gRNAs in conjunction with our light activated CRISPR/dCas9 effector system (LACE). More information about the LACE system can be found on the design page of our wiki (https://2019.igem.org/Team:UC_Davis/Description).
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.
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| 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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 431
Illegal XbaI site found at 873
Illegal PstI site found at 416
Illegal PstI site found at 858
Illegal PstI site found at 1301 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 416
Illegal PstI site found at 858
Illegal PstI site found at 1301 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 431
Illegal XbaI site found at 873
Illegal PstI site found at 416
Illegal PstI site found at 858
Illegal PstI site found at 1301 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 431
Illegal XbaI site found at 873
Illegal PstI site found at 416
Illegal PstI site found at 858
Illegal PstI site found at 1301 - 1000COMPATIBLE WITH RFC[1000]
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