Team:Hong Kong HKUST/riboregulator/future work

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<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/future_work">Future Work</a></li>
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<h2>Riboregulator Future Work</h2>
<h2>Riboregulator Future Work</h2>
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<div class='content_1'><h3> Lysis Module </h3>
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Our team designed but was unable to test this module due to the limitation of not being able to work directly with <i>Streptococcus
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                                                Since the number of regulatory RNAs in part registry have been increasing over the years and they have not been well curated, our team wishes to solve this problem by creating a list of category tags and guidelines so that one can search these parts efficiently. Therefore, we will be continuing our work in testing and characterizing the regulatory RNAs for the ease of the synthetic biology community's use in the future.
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Pneumoniae</i> (Biosafety level 2) in our lab. This module proposes to kill <i>Streptococcus Pneumoniae</i> upon detection when coupled
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with the detection and regulation modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal.
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<p> Please visit our <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/RNA_devices_catalog">prototype of the catalog page</a> and stay tuned with our updates.</p>
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Amidase (PAL)- cleaves the peptidoglycan between N-acetylmuramic acid and L-alanine
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Lysozyme (CPl-1)- cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine
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<div class='content_1'><h3>Re-characterization of Riboregulator under different genetic context</h3>
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<h5>Fig ? . The lysis Module.</h5>
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<p> The enzymes are tagged with osmY (Washington 2012) via a linker to be exported out of <i>Escherichia coli</i>. Both enzymes have
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very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases.
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Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic
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membrane and ultimate lysis of <i>S. pneumoniae</i>.
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<u>References</u>
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                                                The characterization result of riboregulators were out of our expectation - the taRNAs were unable to efficiently unlock their cognate crRNAs. We now realized that our genetic context used was different from how riboregulators were characterized initially. Expression of TAs were supposed to be driven from high copy plasmids while CRs were supposed to be driven from low copy vectors. Thus, we will progressively change the conditions used to characterize the constructs into a new genetic context. We hope that we can replicate others' results soon.</p><br><br>
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J.M. Loeffler et al &quot;Rapid Killing of <i>Streptococcus pneumoniae</i> with a Bacteriophage Cell Wall Hydrolase&quot; 2001
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J.M. Loeffler et al &quot;Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia&quot; 2003
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J.M. Loeffler et al &quot;Synergistic Lethal Effect of a Combination of Phage Lytic Enzymes with Different Activities on Penicillin-Sensitive
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and -Resistant <i>Streptococcus pneumoniae</i> Strains&quot; 2003
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<div class='content_1'><h3>Characterization of Arabinose inducible pBad/araC promoter (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in high copy plasmid</h3>
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<p>We observed a transfer function with an all-or-none response in the pBad/araC promoter on a low copy plasmid during measurement, which matched the experimental result from that of iGEM 2011 Cambridge team, who also used the same genetic context. We are now speculating that the discrepancy between results from iGEM 2011 Cambridge and Groningen was a result due to different copy number plasmids used in carrying the promoter. However, due to lack of sufficient time and manpower, we were unable to test that out in time. Our future work will involve resumption of this characterization, and hopefully, we can explain the discrepancy observed through a mathematical model.</p><br><br>
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST">Home</a></h4>
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<li class='site_link'><a href="">Team</a></li>
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor">Pneumosensor</a></h4>
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<td class= 'site_map_column'><h4><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator">Riboregulator</a></h4>
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Latest revision as of 23:49, 17 October 2014



Riboregulator Future Work

Catalog Page

Since the number of regulatory RNAs in part registry have been increasing over the years and they have not been well curated, our team wishes to solve this problem by creating a list of category tags and guidelines so that one can search these parts efficiently. Therefore, we will be continuing our work in testing and characterizing the regulatory RNAs for the ease of the synthetic biology community's use in the future.



Please visit our prototype of the catalog page and stay tuned with our updates.

Re-characterization of Riboregulator under different genetic context

The characterization result of riboregulators were out of our expectation - the taRNAs were unable to efficiently unlock their cognate crRNAs. We now realized that our genetic context used was different from how riboregulators were characterized initially. Expression of TAs were supposed to be driven from high copy plasmids while CRs were supposed to be driven from low copy vectors. Thus, we will progressively change the conditions used to characterize the constructs into a new genetic context. We hope that we can replicate others' results soon.



Characterization of Arabinose inducible pBad/araC promoter (BBa_I0500) in high copy plasmid

We observed a transfer function with an all-or-none response in the pBad/araC promoter on a low copy plasmid during measurement, which matched the experimental result from that of iGEM 2011 Cambridge team, who also used the same genetic context. We are now speculating that the discrepancy between results from iGEM 2011 Cambridge and Groningen was a result due to different copy number plasmids used in carrying the promoter. However, due to lack of sufficient time and manpower, we were unable to test that out in time. Our future work will involve resumption of this characterization, and hopefully, we can explain the discrepancy observed through a mathematical model.



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