BLA 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 Beta-lactamase fit in our Biosensor?

In the system we are designing, beta-lactamase serves as a reporter as much as prussian blue ferritin. If enterohemorrhagic DNA is present in the sample, the immobilized TALE will capture it in solution. A mobile TALE, which is linked to BLA, will bind to the target DNA, a sequence in the Stx2. The strip is then washed to remove unbound TALEs and a substrate is added to give the colour output.

Constructs

We retrieved the BLA gene from the backbone of the pSB1A3 plasmid. We had done modifications to 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 BLA. Details on how these procedures were done can be found at our Reporter Journal.

Results

For characterization purposes, we are working on testing our constructs with benzylpenicillin, a substrate that gives a colourimetric and a pH output. In the future, we will also characterize TALE A-linker-beta-lactamase 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 beta lactamase was producing functional beta lactamase. In order to do so, we performed an Ampicillin Survival Assay 1 with E. coli transformed with beta-lactamase. We let it grow overnight, spun it down, and resuspended in fresh LB with ampicillin, chloremphenical, and ampicillin and chloremphenicol and we measured the OD at different time points. This assay allowed us to determine whether the beta lactamase was produced and whether it is functional. Only the bacteria producing the beta lactamase was able to survive in ampicillin, which can be seen with an increase in OD whereas our controls did not, which can be seen with a decrease in OD. (Figure 3).

In addition to that, we have purified our beta-lactamase and our mobile TALE linked to beta-lactamase construct (Figure 4) and we have demonstrated that beta lactamase retained its enzymatic activity for both proteins. We repeated a variation of ampicillin survival assay where we pretreated LB containing ampicillin and chloremphenicol with our purified TALE linked to beta-lactamase. We then cultured bacteria in the treated LB that only carry resistance to chloremphenicol. 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 was able to grow in the presence of our purified construct protein, which means that we are expressing and purifying functional protein which is degrading the ampicillin (figures 5 and 6). Figure 5 shows the OD at 24 hour time point from culturing where Figure 6 shows OD change over time. Both graphs show and increase in OD for cultures pre-treated with our protein demonstrating our protein is functional.

After verifying that TALE A-linker-beta-lactamase 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. 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 linked to beta lactamase present in each sample.(Figure 7).

Therefore, we have built and submitted beta-lactamase both on its own and linked to TALE. We have expressed, and purified, and demonstrated its functionality for both proteins. We can show activity for our mobile TALE linked to beta-lactamase for our sensor in two different ways, colourimetric and cell growth. We have submitted a very robust and good reporter to the registry for future iGEM teams.