β-lactamase 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, both in how it can be attached to proteins as well as the various outputs it can give, making it a useful reporter to characterize and add to the Parts Registry.

How does β-lactamase fit in our Biosensor?

β-lactamase serves as another reporter we explored for our system in parallel to our Prussian Prussian blue ferritin reporter. But unlike the Prussian Blue ferritin system, β-lactamase can also be used for a pH output as well. This would add more versatility to our system, as β-lactamase can add a different type of output which would mean that our system is not only limited to the colourimetric outputs. Additionally, the pH output can be paired with different pH indicators to give a variety of colourimetric outputs. We have characterized this ability with two separate pH indicators, phenol red and bromothymol blue, and were able to demonstrate two completely different colourimetric output. In addition, the output of our system can be scaled by altering the number of fused β-lactamase proteins by exploiting the ferritin nanoparticle. This can be achieved through modifying the number of β-lactamase molecules attached to ferritin, ranging from 24 or 12 depending on whether our ferritin nanoparticle consists of the 12 heavy-light subunit fusions (BBa_K157018),or 24 individual subunits, composed of separate light and heavy subunits. The result is a system that can be scaled by utilizing 24 or 12 β-lactamase proteins, or only 1 Prussian blue ferritin core, or one β-lactamase protein fused to a TALE as an alternate. This adds the elements of versatility, flexibility, and most importantly, modularity to our biosensor and serves a vital basis for our platform.

Constructs

We retrieved the ampR gene from the backbone of the pSB1A3 plasmid. We added a a His-tag was added to the N-terminus of it using a flexible glycine linker (BBa_K157013), allowing purification through Ni-NTA protein purification, as well as the lacI promoter for expression (Figure 3). Additionaly, we modified this gene to make it a more useful part for the registry, such as the removal of a BsaI cut-site, making it viable for Golden Gate assembly (Figure 4). We also fused the ampR gene to on our TALE A to bind to our target sequence (Figure 5). This could be used in conjunction with another TALE to act in our strip assay. These modifications resulted in the products shown below:

Results

As a preliminary test to confirm proper protein expression, we tested purified β-lactamase with benzylpenicillin, a substrate that gives a colourimetric and a pH output. First, we wanted to demonstrate that our bacteria carrying the ampR gene was expressing functional β-lactamase. BBa_K1189007. In order to do so, we performed an ampicillin survival assay using E. coli transformed with a plasmid encoding the ampR gene. This assay would involve culturing the bacteria and then exposing them to ampcillin, and survival was then measured by OD. This then 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 β-lactamase enzyme were unable to survive, seen by a decrease in OD. (Figure 6). Therefore, we are able to produce functional β-lactamase enzyme (Figure 6).

After confirming protein expresison, we wanted to demonstrate that we can also purify our proteins. Figure 7 demonstrates that we were able to purify both our β-lactamase (BBa_K1189007) and our TALE-A-β-lactamase protein (BBa_K1189031) (Figure 7). After purification, we wanted to demonstrate whether our purified TALE A - β-lactamase fusion protein retained its enzymatic activity. This was tested by a variation of the 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 carrying the psB1C3 (pSB1C3), conveying resistance to chloramphenicol. In order for the bacteria to survive our isolated protein needed to retain its enzymatic abilities. We can show that the bacteria susceptible to ampicillin were able to grow in the presence of our purified proteins (BBa_K1189031), which means that we are expressing and purifying functional fusion protein which is degrading the ampicillin (Figures 8). 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 pH assay using benzylpenicillin as our substrate to demonstrate that our fusion TALE A - β-lactamase protein can act as a reporter. β-lactamase hydrolyzes benzylpenicillin to penicillinoic acid, which changes the pH of the solution from alkaline to acidic. We tested it out with two separate pH indicators, phenol red and bromothymol blue, and we were able to show two different colourimetric outputs. We were able to show successful reporter acitivy with both indicators.

We were able to see a colour change due to the presence of phenol red, a pH indicator with a transition pH of 6.8-8.2, turning red at lower pH. 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 coincides with the amount of purified TALE A-β-lactamase present in each sample (Figure 10). Additionally, we have demonstrated the same pH change to cause the bromothymol blue to change in colour from blue to yellow as it gets more acidic (Figure 11 & 12). We have demonstrated the reporter activity both qualitatively (Figure 10 & 11) and quantitatively (Figure 12)

Therefore, we have built and submitted parts containing β-lactamase, both on its own and linked to TALE A. We then expressed, purified and demonstrated the final purified products have retained their enzymatic activity. We can show activity for our mobile TALE A linked to β-lactamase (BBa_K1189031) for our sensor in two different ways, through pH and cell growth assays.

Future Directions