It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or DVK_AE (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling.(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)
