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Team:Calgary/Project/OurSensor/Reporter/BetaLactamase
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| - | <p>Many advantages come from working with β-lactamase. It shows high catalytic efficiency and simple kinetics. Also, no orthologs of <i>amp</i>R are known to be encoded by eukaryotic cells and no toxicity was identified making this protein very useful in studies involved eukaryotes (Qureshi, 2007). β-lactamase has been used to track pathogens in infected murine models (Kong <i>et al.</i>, 2010). However, in addition to its application in eukaryotic cells, β-lactamase efficiently cleaves a wide variety of substrates but its versatility goes beyond that; <i>amp</i>R preserves its activity even when fused to heterologous protein (Moore <i>et al.</i>, 1997). This feature, in particular, makes β-lactamase a | + | <p>Many advantages come from working with β-lactamase. It shows high catalytic efficiency and simple kinetics. Also, no orthologs of <i>amp</i>R are known to be encoded by eukaryotic cells and no toxicity was identified making this protein very useful in studies involved eukaryotes (Qureshi, 2007). β-lactamase has been used to track pathogens in infected murine models (Kong <i>et al.</i>, 2010). However, in addition to its application in eukaryotic cells, β-lactamase efficiently cleaves a wide variety of substrates but its versatility goes beyond that; <i>amp</i>R preserves its activity even when fused to heterologous protein (Moore <i>et al.</i>, 1997). This feature, in particular, makes β-lactamase a potentially valuable tool for assembly of synthetic constructs.</p> |
<h2>How is β-lactamase used as a Reporter?</h2> | <h2>How is β-lactamase used as a Reporter?</h2> | ||
<p>β-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 enzymatic activity can be detected by a fluorometer (Remy <i>et al</i>., 2007).</p> | <p>β-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 enzymatic activity can be detected by a fluorometer (Remy <i>et al</i>., 2007).</p> | ||
Revision as of 21:06, 26 October 2013
β-Lactamase
β-Lactamase
What is β-lactamase?
β-lactamase is an enzyme encoded by the ampicillin resistance gene (ampR) frequently present in plasmids for selection. Structurally, β-lactamase is a 29 kDa monomeric enzyme (Figure 1). Its enzymatic activity provides resistance to β-lactam antibiotics such as carbapenems, penicillin and ampicillin through hydrolysis of the β-lactam ring, a structure shared by the β-lactam class of antibiotics (Qureshi, 2007).
Figure 1. 3D structure of β-lactamase obtained from our team’s work in Autodesk Maya. To learn more about our modeling, click here.
Many advantages come from working with β-lactamase. It shows high catalytic efficiency and simple kinetics. Also, no orthologs of ampR are known to be encoded by eukaryotic cells and no toxicity was identified making this protein very useful in studies involved eukaryotes (Qureshi, 2007). β-lactamase has been used to track pathogens in infected murine models (Kong et al., 2010). However, in addition to its application in eukaryotic cells, β-lactamase efficiently cleaves a wide variety of substrates but its versatility goes beyond that; ampR preserves its activity even when fused to heterologous protein (Moore et al., 1997). This feature, in particular, makes β-lactamase a potentially valuable tool for assembly of synthetic constructs.
How is β-lactamase used as a Reporter?
β-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 enzymatic activity can be detected by a fluorometer (Remy et al., 2007).
Besides fluorescence assays, β-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 β-lactamase, which causes a colour change from yellow to red.
ampR 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. β-lactamase serves as a reporter element in our system.
If enterohemorrhagic DNA is present in the sample, the immobilized TALE B will capture it in solution. A mobile TALE A, which is linked to ampR, 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 ampR from the backbone of the
pSB1A3
plasmid. We modified 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 ampR. We have built these constructs: Figure 3. On the left, part
BBa_K1189009. We added a His-tag to β-lactamase to facilitate purification. On the right, part BBa_K1189007. In addition to the His-tag, PLacI + RBS were added upstream of the β-lactamase gene so we can express and characterize our part. Figure 4. Part
BBa_K1189008. We removed the BsaI cut site in the β-lactamase gene so that it could be used for Golden Gate Assembly. Figure 5. Part BBa_K1189031. This construct works as the mobile detector in our biosensor. TALE A is linked to β-lactamase and if the stx2 gene is present in the strip, our mobile is retained on the strip so β-lactamase can give a colour output in the presence of a substrate. For characterization purposes, we tested the constructs with benzylpenicillin, a substrate that gives a colourimetric and a pH output. In the future, we will also characterize TALE A-linker-β-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 β-lactamase gene were expressing a functional enzyme. (BBa_K1189007) was producing functional β-lactamase. In order to do so, we performed an
ampicillin survival assay
with E. coli transformed with β-lactamase. We let the culture grow overnight, then pelleted the cells. We then removed the supernatant and resuspended the cells 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 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 #946;-lactamase enzyme were unable to survive, seenby 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 β-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 β-lactamase (BBa_K1189007) and our mobile TALE A linked to β-lactamase construct (BBa_K1189031) (Figure 7) and we have demonstrated that β-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 β-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 were able to grow in the presence of our purified 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 an increase in OD for cultures pre-treated with our protein demonstrating our protein is functional. Figure 7. On the left crude lysate of β-lactamase + His (BBa_K1189007) from different lysis protocols: a
mechanical
and with sucrose, respectively. On the right, western blot of TALE A-linker-β-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-β-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-β-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-β-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. β-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 β-lactamase present in each sample (Figure 10). Figure 10. Benzylpenicillin assay. On the top, the wells only had
TALE A-linker-β-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 β-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 β-lactamase (BBa_K1189031) for our sensor in two different ways, colourimetric and with cell growth. We feel we have submitted a multi-use reporter to the registry for future iGEM teams to use.
How does β-lactamase fit in our Biosensor?
Constructs
Results
