Comparison with NEU China 2016
Previously, the NEU China 2016 iGEM team characterized their own version of the LACE system. NEU’s version consists of a CRY2 protein linked to a VP64 transcriptional activator domain. We sought out to improve the efficiency of their LACE system by using and characterizing our own modified VP64. The main difference being found in the linker sequence. Our CRY2-VP64 has an extra protein sequence, PIAGSKAS, in between those two genes.
To test which activator was better, we transiently cotransfected cells with the CIBN-dCas9-CIBN and either NEU’s CRY2-VP64, our CRY2-VP64, or CRY2-VPR. 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), which illuminated the wells with blue light for the next 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. A further description of our qPCR analysis can be found on our Experiments page.
