Design

Designing the Gene Insert

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):

• pConst-(RBS)-LuxR-(stop)-prmA-(RBS)-LuxI-(stop)-pLux-(RBS)-GFP-LuxI-(stop)
• pConst-(RBS)-LuxR-(stop)-prmA-(RBS)-LuxI-(stop)-pLux-(RBS)-GFP-(stop)
• pConst-(RBS)-LuxR-(stop)-Lac-(RBS)-LuxI-(stop)-pLux-(RBS)-GFP-LuxI-(stop)
• pConst-(RBS)-LuxR-(stop)-Lac-(RBS)-LuxI-(stop)-pLux-(RBS)-GFP-(stop)

Here is a description of the roles of each of the parts of the insert:

• pConst promoter - Constitutive promoter, constantly stays on and activates LuxR expression, allowing for excess amounts of LuxR in the cell
• LuxR - Protein that binds to 3OC6-HSL (the product of LuxI), creating a complex that then activates the pLux promoter, is created in excess in the cell due to the constitutive promoter. The complex created by LuxR helps with amplification and increased GFP production
• prmA promoter - Turned on by stress in the cell caused by PFAS, expresses LuxI in the gene circuit. This promoter is the main component of our gene insert, and the signal it creates is then amplified by LuxI
• LuxI - Creates quorum sensing molecule (3OC6-HSL) from readily available compounds in the cell, this molecule binds with LuxR to activate the pLux promoter. This step of the gene insert allows for amplification of the prmA signal induced by PFAS
• pLux promoter - Turned on by LuxR:3OC6-HSL complex, activates GFP in the construct and allows for cell fluorescence
• pLac promoter - Found in inserts 3 and 4, used as a control for the experiment. Lac won’t react to PFAS exposure, but by activating the Lac promoter, we would be able to test the efficiency of the rest of the circuit.
• GFP - codes for green fluorescent protein
• RBS - Ribosomal Binding Sites
• Stop - Double terminator sequences, stops transcription

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:

• Part A - pConst-(RBS)-LuxR-(stop)
• Part B - prmA/Lac-(RBS)-LuxI-(stop)
• Part C - pLux-(RBS)-GFP-(stop)

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.