Our Gene Insert Design.
In order to tackle the problem of PFAS, we first had to create a gene circuit that would allow for bacteria to detect PFAS. We created a gene circuit that produces secondary messengers (using quorum sensing molecule 3OC6HSL) in response to PFAS which then increases fluorescent protein expression by inducing a promoter sensitive to QS molecules. We had four possible gene inserts in mind (inserts 3 and 4 contain the Lac promoter as a control for our prmA promoter):
Here is a description of the roles of each of the parts of the insert:
Originally, we were keen on using gene inserts 1 and 3 because of the positive feedback loop created with the addition of the LuxI gene under the influence of the pLux that would be activated by the LuxR:3OC6-HSL complex. This would cause additional synthesis of LuxI, leading to quorum sensing molecules and allowing for more of the LuxR:3OC6-HSL complexes to be formed to reactivate the pLux promoter. However, we decided against this because, since this was our first iteration, we wanted to keep the circuit simpler and see if the insert would still be effective in PFAS detection.
While printing our DNA insert from Twist, we split it into three parts due to fragment length restrictions:
Additionally, while ordering these Parts from Twist, we flanked them on either side with restriction sites to allow for the ligation of the parts in the future. Specifically, the restriction sites added were for the enzymes EcoRI, XBal, SpeI, and PstI (all produce sticky ends). Note that the sticky ends for Xbal and SpeI are compatible. The theoretical ligation procedure is as follows:
Theoretically with the final insert, the plasmid and the insert could both be digested with EcoRI and PstI in order to ligate the fragment into a plasmid vector which could then be transformed into a bacteria cell. This ligation plan was adapted from iGEM’s 3A assembly method, except this procedure aims to ligate 3 fragments instead of 2.
If the assembly goes to plan, a gel electrophoresis of our assembled insert should show a band around 3000 bp.