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Team:Calgary/Project/OurSensor/Reporter/BetaLactamase
From 2013.igem.org
Beta-Lactamase
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).
Figure 1. 3D structure of beta-lactamase obtained from our team’s work in Autodesk Maya. To learn more about our modeling, click here.
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 assembly 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 yellow to red (Remy et al., 2007). Colourimetric assays can also be done with benzylpenicillin as the substrate, which, gives a pH output that can be detected with pH indicators to give a colourimetric output (Li et al., 2008).
Figure 2. Hydrolysis of nitrocefin catalyzed beta-lactamase, which causes a colour change from yellow to red..
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. 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 A will capture it in solution. A mobile TALE A, which is linked to BLA, 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. 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. Figure 3. On the left, part
BBa_K1189009
. We added a His-tag to beta-lactamase to facilitate purification. On the right, part
BBa_K1189007
. In addition to the His-tag, pLacI + RBS were added upstream of the beta-lactamase gene so we can express and characterize our part. Figure 4. Part
BBa_K1189008
. We removed the BsaI cut site in the beta-lactamase gene so we could use it for Golden Gate Assembly. Figure 5. Part
BBa_K1189031
. This construct works as the mobile detector in our biosensor. TALE A is linked to beta-lactamase and if the Stx2 gene is present in the strip, our mobile is retained on the strip so beta-lactamase can give a colour output in the presence a substrate. 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 (
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 beta-lactamase (
BBa_K1189007
) 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, chloramphenicol, and ampicillin and chloramphenicol 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 6). Figure 6. 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 7) 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 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 and increase in OD for cultures pre-treated with our protein demonstrating our protein is functional. Figure 7. 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 8. 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 9. 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 10). Figure 10. 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.
Therefore, we have built and submitted beta-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 beta-lactamase (
BBa_K1189031
) 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.
How does Beta-lactamase fit in our Biosensor?
Constructs
Results
