ampR can also be split apart in to two halves for protein complementation assays where each half is linked to one of the two proteins being tested. If the two proteins interact the two halves are able to fold into their correct structure and give an output (Wehrman et al., 2002).

Therefore, this enzyme gives a lot of flexibility in terms of how it can be used in a system, which makes it a useful reporter to characterize and add to the Parts Registry.

How does β-lactamase fit in our Biosensor?

β-lactamase serves as a reporter element in our system. If enterohemorrhagic DNA is present in the sample, the immobilized TALE B will capture it in solution. A mobile TALE A, which is linked to ampR, will bind to the target DNA, a sequence in the stx2. The strip is then washed to remove unbound TALE A and a substrate is added to give the colour output.

Constructs

We retrieved ampR from the backbone of the pSB1A3 plasmid. We modified it to improve the part such as removal of a BsaI cut site, addition of a His-tag and fusion to a flexible glycine linker (BBa_K157013) to the N-terminus of ampR. We have built these constructs:

Results

For characterization purposes, we tested the constructs with benzylpenicillin, a substrate that gives a colourimetric and a pH output. In the future, we will also characterize TALE A-linker-β-lactamase (BBa_K1189031) in the presence of a nitrocefin which is the substrate we plan to use in our Biosensor.

First, we wanted to demonstrate that our bacteria carrying the β-lactamase gene were expressing a functional enzyme. (BBa_K1189007) was producing functional β-lactamase. In order to do so, we performed an ampicillin survival assay with E. coli transformed with β-lactamase. We let the culture grow overnight, then pelleted the cells. We then removed the supernatant and resuspended the cells in fresh LB with ampicillin, chloramphenicol, and ampicillin and chloramphenicol and we measured the OD at different time points. This assay allowed us to determine whether the β-lactamase was produced and whether it is functional. Only the bacteria producing functional β-lactamase enzymes were able to survive in the presence of ampicillin resulting in an increase in OD. Whereas bacteria lacking the abililty to produce functional #946;-lactamase enzyme were unable to survive, seenby a decrease in OD. (Figure 6).

In addition to that, we have purified our β-lactamase (BBa_K1189007) and our mobile TALE A linked to β-lactamase construct (BBa_K1189031) (Figure 7) and we have demonstrated that β-lactamase retained its enzymatic activity for both proteins. We repeated a variation of ampicillin survival assay where we pretreated LB containing ampicillin and chloramphenicol with our purified TALE A linked to β-lactamase (BBa_K1189031). We then cultured bacteria in the treated LB that only carry resistance to chloramphenicol. Therefore, the bacteria are only able to survive if the our isolated protein retained its enzymatic abilities. We can show that the bacteria susceptible to ampicillin were able to grow in the presence of our purified protein (BBa_K1189031), which means that we are expressing and purifying functional protein which is degrading the ampicillin (Figures 6 and 8). Figure 8 shows the OD at 24 hour time point from culturing where Figure 6 shows OD change over time. Both graphs show an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional.

After verifying that TALE A-linker-β-lactamase (BBa_K1189031) retained enzymatic activity and was able to degrade ampicillin, we performed a colourimetric assay using benzylpenicillin as our substrate. We were able to see a colour change from red to yellow. This is because there is phenol red, a pH indicator, added to the substrate solution. β-lactamase hydrolyzes benzylpenicillin to penicillinoic acid, which changes the pH of the solution from alkaline to acidic. This pH change causes the phenol red to change from red to yellow. Our negative controls, to which benzylpenicillin was not added, remained red. We can also see the colour change correlate to the amount of purified TALE A linked to β-lactamase present in each sample (Figure 10).

Therefore, we have built and submitted β-lactamase both on its own and linked to TALE A. We have expressed, and purified, and demonstrated its functionality for both proteins. We can show activity for our mobile TALE A linked to β-lactamase (BBa_K1189031) for our sensor in two different ways, colourimetric and with cell growth. We feel we have submitted a multi-use reporter to the registry for future iGEM teams to use.