Our plasmids contain GFP coding device and RFP coding device, in varying orientations, with either a 500bp spacer, 1000bp spacer, or a dcas9 clamp binding site between them. They are on a vector with either Chloramphenicol or Kanamycin resistance. (For more details, see Design page —insert hyperlink!) After creating successful plasmids, we then transformed them in E. coli. The colonies were then inoculated and grown as overnight liquid cultures.
Experiment:
Plate Reader~
For in vivo using liquid cultures:
For cultures that contained colonies with plasmids that had a 500bp spacer or 1000bp spacer, we diluted these cultures to normalize them to the same OD absorbance. Then, we ran these in the plate reader to measure RFP (in AFU), GFP (in AFU), and OD (abs). For culture using a 1000bp spacer we tried testing the effects of using inducer as well. Using these results we were able to analyze the effects of supercoiling and the effects of using a base-pair spacer between them in order to reduce supercoiling. Because the GFP and RFP devices are placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (see Plate Reading protocol in Protocols: https://2016.igem.org/Team:Alverno_CA/Protocols)
For in vitro using liquid plasmid DNA by TX-TL:
For cultures that contained colonies that had a dcas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. We also designed and gRNA plasmids to use for testing as well and got dcas9 expression plasmids (DS-SPCasN- —insert hyperlink to AddGene!) from Caltech’s Murray Lab. All three of these plasmids were then expressed by in vitro using TX-TL. For our first run we did not use inducer, but for our second run we used ATc and IPTG to induce the TetR and LacI promoters on the GFP and RFP devices, respectively. After running this by TX-TL in the plate reader we obtained results that we then analyzed to determine whether or not supercoiling was reduced and GFP and RFP expressed. Once again, because the GFP and RFP devices are placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (see Plate Reading protocol in)
