Part:BBa_K1189008
Beta-Lactamase
Beta-lactamase (BLA) is an enzyme frequently present in plasmids for selection. Beta-lactamase is a 29-kDa monomeric enzyme. 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). Kinetically, beta-lactamase is simple and shows high catalytic efficiency. 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). BLA has also 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. This is submitted in the Freiburg fusion backbone for easy fusion construction. Additionally, the BsaI cut site in the gene has been mutagenized.
Figure 1. Absorbance values at 600nm for each tube at four different time points: 0, 30, 60 and 120min. The cultures that expressed beta-lactamase (
BBa_K1189007
) showed higher absorbance levels, showing that the cells were able to grow in the presence of ampicillin.
In addition to that, we have purified our beta-lactamase (
BBa_K1189007
) and our mobile TALE A linked to beta-lactamase construct (
BBa_K1189031
) (Figure 2) 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 chloramphenicol with our purified TALE A linked to beta-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 was able to grow in the presence of our purified construct protein (
BBa_K1189031
), which means that we are expressing and purifying functional protein which is degrading the ampicillin (Figures 1 and 3). Figure 3 shows the OD at 24 hour time point from culturing where Figure 1 shows OD change over time. Both graphs show an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional. Figure 2. On the left crude lysate of beta-lactamase + His (
BBa_K1189007
) from different lysis protocols: a
mechanical
and with
sucrose
, respectively. On the right, western blot of
TALE A
-linker-beta-lactamase (
BBa_K1189031
) showing that we were able to express and purify our construct.
Figure 3. Absorbance values at 600nm after 24h. Amounts from 0.1µg to 20µg of TALE A-link-Beta-lactamase (
BBa_K1189031
) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.
Figure 4. Absorbance values at 600nm in different time points. Amounts from 1.0µg to 10µg of TALE A-link-Beta-lactamase (
BBa_K1189031
) were sufficient to degrade the ampicillin in the media allowing bacteria susceptible to ampicillin to grow.
After verifying that
TALE A
-linker-beta-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. Beta-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 beta-lactamase present in each sample (Figure 5). Figure 5. Benzylpenicillin assay. On the top, the wells only had
TALE A
-linker-beta-lactamase (
BBa_K1189031
). Benzylpenicillin was added and after a 10-minute incubation at room temperature, we were able to observe a colour output from red to yellow (bottom row) while the control wells remained red.
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