Team:Calgary/Project/OurSensor/Reporter/BetaLactamase
From 2013.igem.org
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- | <h1> | + | <h1>β-Lactamase</h1> |
<h2>What is β-lactamase?</h2> | <h2>What is β-lactamase?</h2> | ||
- | <p> | + | <p>β-lactamase is an enzyme encoded by the ampicillin resistant gene (<i>amp</i>R) 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 cephamysin, carbapenems and penicillium through hydrolysis of the β-lactam ring, a structure shared by these antibiotics (Qureshi, 2007).</p> |
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/a/ad/UCalgary2013TRBetalactamaserender.png"> | <img src="https://static.igem.org/mediawiki/2013/a/ad/UCalgary2013TRBetalactamaserender.png"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 1.</b> 3D structure of | + | <p><b>Figure 1.</b> 3D structure of β-lactamase obtained from our team’s work in Autodesk Maya. To learn more about our modeling, click <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Modeling"><span class="Green"><b>here</b></span></a>. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
- | <p>Many advantages come from working with | + | <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 potential tool for assembly of synthetic constructs.</p> |
- | <h2>How is | + | <h2>How is β-lactamase used as a Reporter?</h2> |
- | <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> |
- | <p>Besides fluorescence assays, | + | <p>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 <i>et al</i>., 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 <i>et al</i>., 2008). |
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/7/7f/YYC2013_Blac_Nitrocefin.jpg"> | <img src="https://static.igem.org/mediawiki/2013/7/7f/YYC2013_Blac_Nitrocefin.jpg"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 2.</b> Hydrolysis of nitrocefin catalyzed | + | <p><b>Figure 2.</b> Hydrolysis of nitrocefin catalyzed β-lactamase, which causes a colour change from yellow to red.</a> |
</figcaption> | </figcaption> | ||
<p><i>amp</i>R 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 <i>et al.</i>, 2002).</p> | <p><i>amp</i>R 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 <i>et al.</i>, 2002).</p> | ||
<p>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.</p> | <p>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.</p> | ||
- | <h2>How does | + | <h2>How does β-lactamase fit in our Biosensor?</h2> |
- | <p> | + | <p>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 <i>amp</i>R, will bind to the target DNA, a sequence in the <i>stx2</i>. The strip is then washed to remove unbound TALE A and a substrate is added to give the colour output.</p> | 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 <i>amp</i>R, will bind to the target DNA, a sequence in the <i>stx2</i>. The strip is then washed to remove unbound TALE A and a substrate is added to give the colour output.</p> | ||
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BBa_K1189009 | BBa_K1189009 | ||
</b></span> | </b></span> | ||
- | </a>. We added a His-tag to | + | </a>. We added a His-tag to β-lactamase to facilitate purification. On the right, part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189007"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189007 | BBa_K1189007 | ||
</b></span> | </b></span> | ||
- | </a>. In addition to the His-tag, PLacI + RBS were added upstream of the | + | </a>. In addition to the His-tag, PLacI + RBS were added upstream of the β-lactamase gene so we can express and characterize our part.</p> |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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BBa_K1189008 | BBa_K1189008 | ||
</b></span> | </b></span> | ||
- | </a>. We removed the BsaI cut site in the | + | </a>. We removed the BsaI cut site in the β-lactamase gene so that it could be used for Golden Gate Assembly.</p> |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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BBa_K1189031 | BBa_K1189031 | ||
</b></span> | </b></span> | ||
- | </a>. This construct works as the mobile detector in our biosensor. TALE A is linked to | + | </a>. This construct works as the mobile detector in our biosensor. TALE A is linked to β-lactamase and if the <i>stx2</i> 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.</p> |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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TALE A | TALE A | ||
</b></span> | </b></span> | ||
- | </a>-linker- | + | </a>-linker-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
</b></span> | </b></span> | ||
</a >) in the presence of a nitrocefin which is the substrate we plan to use in our Biosensor.</p> | </a >) in the presence of a nitrocefin which is the substrate we plan to use in our Biosensor.</p> | ||
- | <p> First, we wanted to demonstrate that our bacteria carrying the | + | <p> First, we wanted to demonstrate that our bacteria carrying the β-lactamase gene were expressing a functional enzyme. (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189007"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189007 | BBa_K1189007 | ||
</b></span> | </b></span> | ||
- | </a>) was producing functional | + | </a>) was producing functional β-lactamase. In order to do so, we performed an <a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/AmpicillinSurvivalAssay1"> |
<span class="Green"><b> | <span class="Green"><b> | ||
Ampicillin Survival Assay 1 | Ampicillin Survival Assay 1 | ||
</b></span> | </b></span> | ||
- | </a> with <i>E. coli</i> transformed with | + | </a> with <i>E. coli</i> 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 the β-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).</p> |
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/thumb/0/03/YYC2013_Blac_Amp_Survival_Assay_with_colonies.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_colonies.jpg"> | <img src="https://static.igem.org/mediawiki/2013/thumb/0/03/YYC2013_Blac_Amp_Survival_Assay_with_colonies.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_colonies.jpg"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 6. </b>Absorbance values at 600nm for each tube at four different time points: 0, 30, 60 and 120min. The cultures that expressed | + | <p><b>Figure 6. </b>Absorbance values at 600nm for each tube at four different time points: 0, 30, 60 and 120min. The cultures that expressed β-lactamase (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189007"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189007 | BBa_K1189007 | ||
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</a>) showed higher absorbance levels, showing that the cells were able to grow in the presence of ampicillin.</a> | </a>) showed higher absorbance levels, showing that the cells were able to grow in the presence of ampicillin.</a> | ||
</figcaption> | </figcaption> | ||
- | <p>In addition to that, we have purified our | + | <p>In addition to that, we have purified our β-lactamase (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189007"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189007 | BBa_K1189007 | ||
</b></span> | </b></span> | ||
- | </a>) and our mobile TALE A linked to | + | </a>) and our mobile TALE A linked to β-lactamase construct (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
</b></span> | </b></span> | ||
- | </a >) (Figure 7) and we have demonstrated that | + | </a >) (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 (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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<img src="https://static.igem.org/mediawiki/2013/4/45/YYC2013_TALE_September_22_Blac.jpg"> | <img src="https://static.igem.org/mediawiki/2013/4/45/YYC2013_TALE_September_22_Blac.jpg"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 7. </b>On the left crude lysate of | + | <p><b>Figure 7. </b>On the left crude lysate of β-lactamase + His (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189007"> |
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BBa_K1189007 | BBa_K1189007 | ||
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TALE A | TALE A | ||
</b></span> | </b></span> | ||
- | </a>-linker- | + | </a>-linker-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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<img src="https://static.igem.org/mediawiki/2013/thumb/3/38/YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg"> | <img src="https://static.igem.org/mediawiki/2013/thumb/3/38/YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_24h.jpg"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 8. </b>Absorbance values at 600nm after 24h. Amounts from 0.1µg to 20µg of TALE A-link- | + | <p><b>Figure 8. </b>Absorbance values at 600nm after 24h. Amounts from 0.1µg to 20µg of TALE A-link-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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<img src="https://static.igem.org/mediawiki/2013/thumb/d/de/YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg"> | <img src="https://static.igem.org/mediawiki/2013/thumb/d/de/YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg/800px-YYC2013_Blac_Amp_Survival_Assay_with_protein_3_time_points.jpg"> | ||
<figcaption> | <figcaption> | ||
- | <p><b>Figure 9. </b>Absorbance values at 600nm in different time points. Amounts from 1.0µg to 10µg of TALE A-link- | + | <p><b>Figure 9. </b>Absorbance values at 600nm in different time points. Amounts from 1.0µg to 10µg of TALE A-link-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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TALE A | TALE A | ||
</b></span> | </b></span> | ||
- | </a>-linker- | + | </a>-linker-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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colourimetric assay | colourimetric assay | ||
</b></span> | </b></span> | ||
- | </a> 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. | + | </a> 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).</p> |
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2013/8/86/YYC2013_Blac_%2B_Penicillium_G.jpg"> | <img src="https://static.igem.org/mediawiki/2013/8/86/YYC2013_Blac_%2B_Penicillium_G.jpg"> | ||
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TALE A | TALE A | ||
</b></span> | </b></span> | ||
- | </a>-linker- | + | </a>-linker-β-lactamase (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 | ||
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</figcaption> | </figcaption> | ||
- | <p> Therefore, we have built and submitted | + | <p> 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 (<a href=" http://parts.igem.org/wiki/index.php?title=Part:BBa_K1189031"> |
<span class="Green"><b> | <span class="Green"><b> | ||
BBa_K1189031 | BBa_K1189031 |
Revision as of 22:30, 23 October 2013
β-Lactamase
β-Lactamase
What is β-lactamase?
β-lactamase is an enzyme encoded by the ampicillin resistant 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 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 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 potential 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).