We believed that supercoiling has been a problem for synthetic biologists trying to construct plasmids expressing multiple genes. We created plasmids to demonstrate supercoiling and the interference it creates between two genes. Our plasmids contain one GFP coding device and one RFP coding device, in varying orientations, with either a 500bp spacer, 1000bp spacer, or dCas9 clamp binding sites between them. The plasmids use the backbone pSB1C3 or DVK_AE (derived from pSB1K3). 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. (For more details, see Design) After creating successful plasmids, we 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 containing strains with plasmids that had a 500bp spacer or 1000bp spacer, we diluted these cultures to normalize them to the same OD absorbance. Then, we measured RFP expression (in AFU), GFP expression(in AFU), and OD (absorbance) in a plate reader (VICTOR-X3). For strains using a 1000bp spacer we tried testing the effects of using inducer (ATc and IPTG) 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. (See our Plate Reading (for Fluorescence, Absorbance, Induction, etc.) Protocol for more information)
For in vitro using liquid plasmid DNA by TX-TL
For cultures containing strains with plasmids that had a dCas9 clamp site spacer, cultures were mini-prepped to extract the plasmid DNA. In order to run our plasmids containing the dCas9 clamp site, we also had to design gRNA plasmids and obtain dCas9 expression plasmids. (DS-SPCasN-)). All three of these plasmids were miniprepped and then expressed using TX-TL, an in vitro, prototyping technique which mimics cell environments for transcription and translation. 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. In the plate reader we obtained results that measured GFP and RFP expression. (see Results) Once again, because the GFP and RFP devices were placed in different orientations we can observe whether or not supercoiling is present and whether or not it was reduced. (See our Plate Reading (for Fluorescence using TX-TL for GG105-108 w/ dcas9 expression plasmids) Protocol for more information)
