Beta-Lactamase

What is Beta-lactamase?

Beta-lactamase (BLA) is an enzyme encoded by the ampicillin resistant gene (ampr) frequently present in plasmids for selection. Structurally, beta-lactamase is a 29-kDa monomeric enzyme (Figure 1). Its enzymatic activity provides resistance to beta-lactam antibiotics such as cephamysin, carbapenems and penicillium through hydrolysis of the β-lactam ring, a structure shared by these antibiotics (Qureshi, 2007).

Many advantages come from working with beta-lactamase. It shows high catalytic efficiency and simple kinetics. Also, no orthologs of BLA are known to be encoded by eukaryotic cells and no toxicity was identified making this protein very useful in studies involved eukaryotes (Qureshi, 2007). Beta-lactamase has been used to track pathogens in infected murine models (Kong et. al, 2010). However, in addition to its application in eukaryotic cells, beta-lactamase efficiently cleaves a wide variety of substrates but its versatility goes beyond that; BLA preserves its activity even when fused to heterologous protein (Moore et. al, 1997). This feature, in particular, makes beta-lactamase a potential tool for assemble of synthetic constructs.

How is Beta-lactamase used as a Reporter?

Beta-lactamase, in the presence of different substrates, can give various outputs. It can produce a fluorogenic output in the presence of a cephalosporin derivative (CCF2/AM) and BLA enzymatic activity can be detected by a fluorometer (Remy et al., 2007).

Besides fluorescence assays, beta-lactamase can also be used to obtain colourimetric outputs by breaking down synthetic compounds such as nitrocefin (Figure 2). The colour change goes from red to yellow (Remy et al., 2007). Colourimetric assays can also be done with penicillin G as the substrate, which, gives a pH output that can be detected with pH indicators to give a colourimetric output (Li et al., 2008).

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 removed the BsaI cut site so it can be used for Golden Gate Assembly and we also added a His-tag to it. Another modification includes fusion of a flexible glycine linker (BBa_K157013) to the N-terminus of BLA so we could later connect it to our detector. More 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 penicillium G, 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.

We have performed an Ampicillin Survival Assay 1 with E. coli transformed with beta-lactamase and we measured the OD in different time points and we verified that the bacteria was able to grow in LB + amp, which means that it is able to express beta-lactamase (Figure 3).

In addition to that, we have purified our mobile TALE linked to beta-lactamase construct (Figure 4) and we have demonstrated that BLA retained its enzymatic activity. Bacteria susceptible to ampicillin was able to grow in the presence of our construct, which means it is degrading the antibiotic (figures 5 and 6).

After verifying that TALE A-linker-beta-lactamase retained enzymatic activity and was able to degrade ampicillin, we performed a colourimetric assay using Penicillium G as our substrate. We were able to see a colour change from red to yellow. Our negative controls, to which Penicillium G was not added, remained red (Figure 7).