Difference between revisions of "Team:Warwick/Design"

Revision as of 03:04, 18 October 2018 (view source)

KurtHill (Talk | contribs)

m

← Older edit

Revision as of 03:11, 18 October 2018 (view source)

KurtHill (Talk | contribs)

m

Newer edit →

Line 135:

$("#Content2").html("<h2>Plasmids and Sequences</h2><img src='https://static.igem.org/mediawiki/2018/5/5d/T--Warwick--ribodesign2.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/5/59/T--Warwick--ribodesign3.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/f/f5/T--Warwick--ribodesign4.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/d/d2/T--Warwick--ribodesign5.png'><p>The full annotated plasmids which we used for our experiments are shared for the iGEM community to use. We have highlighted on every plasmid where our Legionella Sensing Lac Repressing and Pathogen Imitating Domains exist. All five iterations of plasmids that we designed are available in genbank format: <a href='http://parts.igem.org/File:T--Warwick--2018-Entire-Project.zip'>here</a><br><br>We have the full plasmid<br><br>gRNA secondary structure prediction<br><br>ATGTTTAAACACGGATGTTTTATACTAATAGTCTTTTTTT TAGCCAGCTGCTTGTCAGGCTGTCTGGGGGTTTTATGGAC TGGGGCAACTCTAGCCTATGACCGGCACAATGTATATAAA AAATTAGATGATTATAATTTAATAAAAGCTCTTAATGATG TTTTAGCTGTCAACCGGACATTTAAAAACTCAGAGACAGT ACTGGATATTGCTGTGTTCAACAGAGATATTTTAATAGCA GGGCATGTTCCAACCCAGGAGTTGTACGATGAATTACAAC TACGTCTAAGCAAAGTTAAAGGATACAGGCGGTTATTCAA TCGTGTAATTATCAATAAAATGCCATCCAACTCGATCCAG GACAGCTGGATTACCACGAAAATCCGTAGCCAAATTTTTG CAGACTCTTCAATCGATCCTAACGCTTTCAAGGTGGTTAC TTCTGATCGTGTTGTCTATTTGATGGGAGATGTTCAGACA GAACAAGCTGAAAAAGTGATTAAAATTGCAAGATACACAG GTGGTGTACAGAAAGTAATAAAACTTATGCGGTATTATAC TTATCAAACCAGTACGAATATGGCCTGA<br><br><br>Initially we determined the <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha riboswitch</a> by reading the DNA sequence and searching for potential loops by many repeated characters or similar bases near one another. The region in bold highlights a potential sense loop domain recognition region and the region in italics shows the extended sensing domain.</p>");

−

$("#Content3").html("<img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/7/77/T--Warwick--ribodesign6.png'><br><p>The Gamma construct was designed by analysing the secondary structure of the Legiolysin mRNA and locating exposed nucleotides. The potential loop recognition domain highlighted in red shows the region that construct Gamma was designed through prediction of exposed nucleotides for the optimal mRNA-gRNA interaction efficiency. We utilised extensive secondary structure prediction through our model ~~[LINK]~~ utilising NuPack to determine functional switch sequences.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/3/33/T--Warwick--ribodesign7.png'><br><p>The <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha construct</a> prediction determined that the Blocking domain effectively obstructed the Targeting Domain. The blocked targeting domain would not function, thus the switch would be in the off state.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/9/94/T--Warwick--ribodesign8.png'><br><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/a/aa/T--Warwick--2018-DNAwhole.gif'><br><p>We used centrifold method of folding with RNA composer to get some <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>results</a></p>");

+

$("#Content3").html("<img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/7/77/T--Warwick--ribodesign6.png'><br><p>The Gamma construct was designed by analysing the secondary structure of the Legiolysin mRNA and locating exposed nucleotides. The potential loop recognition domain highlighted in red shows the region that construct Gamma was designed through prediction of exposed nucleotides for the optimal mRNA-gRNA interaction efficiency. We utilised extensive secondary structure prediction through our model utilising NuPack to determine functional switch sequences.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/3/33/T--Warwick--ribodesign7.png'><br><p>The <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha construct</a> prediction determined that the Blocking domain effectively obstructed the Targeting Domain. The blocked targeting domain would not function, thus the switch would be in the off state.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/9/94/T--Warwick--ribodesign8.png'><br>The Alpha and Pathogen Imitating Sequence complex prediction determined that the Blocking domain was not able to be removed from the Targeting Domain, implying that the switch would remain in the off state regardless of stimulation. Despite the information we experimented with the construct to test the accuracy of the modelling software and found that the model was not representative of results. A large amount of our time was dedicated to <a href='https://2018.igem.org/Team:Warwick/Model'>modelling</a> our constructs until they demonstrated effective function.<br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/a/aa/T--Warwick--2018-DNAwhole.gif'><br><p>We used centrifold method of 3D RNA folding prediction with RNA composer to test the tertiary structure of our constructs with the 2D predicted structures from NuPack. <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>The predicted structures</a> supported our initial conclusions that the targeting domain of the gRNA (green) was inhibited by the blocking domain (red) and that the sensing loop (blue) and extended sensing domain (light blue) could unfold the structure while the dCAS9 recognition cis acting factor (grey) did not disrupt the blocking and targeting domains. Watson-Crick base pairing of the gRNA targeting domain and the blocking domain is clear through analysis of the molecular structure. <br>We used centrifold method of folding with RNA composer to get some <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>results</a></p>");

$("#Content4").html("<h2>Alpha RNA Construct:</h2><br><p>CCCAGACAGCCTGAC AAAGGTTTTGCAC GCTAAAAAAAAGACT GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Alpha RNA Pathogen-Imitating Region</h2><br><p>AGTCTTTTTTTTAGC CAGCTGCTT GTCAGGCTGTCTGGGatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Beta RNA Construct:</h2><br>p>GTACACCACCTGTGTA AAAGGTTTTGCAC TTTTAATCACTTTTT GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Beta RNA Pathogen-Imitating Region</h2><br><p>AAAAAGTGATTAAAA TTGCAAGAT<br>ACACAGGTGGTGTACatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Gamma RNA Construct:</h2><br><p>GGTTGACAGCTAAAA AAAGGTTTTGCAC GAGCTTTTATTAAATTATAATC GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG<br><br><br><h2>Gamma RNA Pathogen-Imitating Region</h2><br><p>GATTATAATTTAATAAAAGCTC TTAATGATG TTTTAGCTGTCAACCatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Delta RNA Construct:</h2><br><p>TAGGCTAGAGTTGCCAAAGGTTTTGCAC AAAACCCCCAGACAG GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Delta RNA Pathogen-Imitating Region</h2><br><p>CTGTCTGGGGGTTTTATGGACTGGGGCAACTCTAGCCTAatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Epsilon RNA Construct:</h2><br><p>TCGAGTTGGATGGCAAAAGGTTTTGCAC TAATTACACGATTGAATAACCGCC GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Epsilon RNA Pathogen-Imitating Region:</h2><br><p>GGCGGTTATTCAATCGTGTAATTATCAATAAAATGCCATCCAACTCGAatccttagcgaaagctaaggatttttttt</p>");

Difference between revisions of "Team:Warwick/Design"

Revision as of 03:11, 18 October 2018

Design

Contact Us

igem@warwick.ac.uk

Sponsors

@@ Line 135: / Line 135: @@
          $("#Content2").html("<h2>Plasmids and Sequences</h2><img src='https://static.igem.org/mediawiki/2018/5/5d/T--Warwick--ribodesign2.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/5/59/T--Warwick--ribodesign3.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/f/f5/T--Warwick--ribodesign4.png'><img style='width: 80%' src='https://static.igem.org/mediawiki/2018/d/d2/T--Warwick--ribodesign5.png'><p>The full annotated plasmids which we used for our experiments are shared for the iGEM community to use. We have highlighted on every plasmid where our Legionella Sensing Lac Repressing and Pathogen Imitating Domains exist. All five iterations of plasmids that we designed are available in genbank format: <a href='http://parts.igem.org/File:T--Warwick--2018-Entire-Project.zip'>here</a><br><br>We have the full plasmid<br><br>gRNA secondary structure prediction<br><br>ATGTTTAAACACGGATGTTTTATACTAATAGTCTTTTTTT TAGCCAGCTGCTTGTCAGGCTGTCTGGGGGTTTTATGGAC TGGGGCAACTCTAGCCTATGACCGGCACAATGTATATAAA AAATTAGATGATTATAATTTAATAAAAGCTCTTAATGATG TTTTAGCTGTCAACCGGACATTTAAAAACTCAGAGACAGT ACTGGATATTGCTGTGTTCAACAGAGATATTTTAATAGCA GGGCATGTTCCAACCCAGGAGTTGTACGATGAATTACAAC TACGTCTAAGCAAAGTTAAAGGATACAGGCGGTTATTCAA TCGTGTAATTATCAATAAAATGCCATCCAACTCGATCCAG GACAGCTGGATTACCACGAAAATCCGTAGCCAAATTTTTG CAGACTCTTCAATCGATCCTAACGCTTTCAAGGTGGTTAC TTCTGATCGTGTTGTCTATTTGATGGGAGATGTTCAGACA GAACAAGCTGAAAAAGTGATTAAAATTGCAAGATACACAG GTGGTGTACAGAAAGTAATAAAACTTATGCGGTATTATAC TTATCAAACCAGTACGAATATGGCCTGA<br><br><br>Initially we determined the <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha riboswitch</a> by reading the DNA sequence and searching for potential loops by many repeated characters or similar bases near one another. The region in bold highlights a potential sense loop domain recognition region and the region in italics shows the extended sensing domain.</p>");
-         $("#Content3").html("<img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/7/77/T--Warwick--ribodesign6.png'><br><p>The Gamma construct was designed by analysing the secondary structure of the Legiolysin mRNA and locating exposed nucleotides. The potential loop recognition domain highlighted in red shows the region that construct Gamma was designed through prediction of exposed nucleotides for the optimal mRNA-gRNA interaction efficiency. We utilised extensive secondary structure prediction through our model [LINK] utilising NuPack to determine functional switch sequences.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/3/33/T--Warwick--ribodesign7.png'><br><p>The <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha construct</a> prediction determined that the Blocking domain effectively obstructed the Targeting Domain. The blocked targeting domain would not function, thus the switch would be in the off state.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/9/94/T--Warwick--ribodesign8.png'><br><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/a/aa/T--Warwick--2018-DNAwhole.gif'><br><p>We used centrifold method of folding with RNA composer to get some <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>results</a></p>");
+         $("#Content3").html("<img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/7/77/T--Warwick--ribodesign6.png'><br><p>The Gamma construct was designed by analysing the secondary structure of the Legiolysin mRNA and locating exposed nucleotides. The potential loop recognition domain highlighted in red shows the region that construct Gamma was designed through prediction of exposed nucleotides for the optimal mRNA-gRNA interaction efficiency. We utilised extensive secondary structure prediction through our model utilising NuPack to determine functional switch sequences.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/3/33/T--Warwick--ribodesign7.png'><br><p>The <a href='http://parts.igem.org/Part:BBa_K2841541'>Alpha construct</a> prediction determined that the Blocking domain effectively obstructed the Targeting Domain. The blocked targeting domain would not function, thus the switch would be in the off state.</p><br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/9/94/T--Warwick--ribodesign8.png'><br>The Alpha and Pathogen Imitating Sequence complex prediction determined that the Blocking domain was not able to be removed from the Targeting Domain, implying that the switch would remain in the off state regardless of stimulation. Despite the information we experimented with the construct to test the accuracy of the modelling software and found that the model was not representative of results. A large amount of our time was dedicated to <a href='https://2018.igem.org/Team:Warwick/Model'>modelling</a> our constructs until they demonstrated effective function.<br><br><img style='width: 80%; background-color: white;' src='https://static.igem.org/mediawiki/2018/a/aa/T--Warwick--2018-DNAwhole.gif'><br><p>We used centrifold method of 3D RNA folding prediction with RNA composer to test the tertiary structure of our constructs with the 2D predicted structures from NuPack. <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>The predicted structures</a> supported our initial conclusions that the targeting domain of the gRNA (green) was inhibited by the blocking domain (red) and that the sensing loop (blue) and extended sensing domain (light blue) could unfold the structure while the dCAS9 recognition cis acting factor (grey) did not disrupt the blocking and targeting domains. Watson-Crick base pairing of the gRNA targeting domain and the blocking domain is clear through analysis of the molecular structure. <br>We used centrifold method of folding with RNA composer to get some <a href='https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703931/'>results</a></p>");
          $("#Content4").html("<h2>Alpha RNA Construct:</h2><br><p>CCCAGACAGCCTGAC AAAGGTTTTGCAC GCTAAAAAAAAGACT GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Alpha RNA Pathogen-Imitating Region</h2><br><p>AGTCTTTTTTTTAGC CAGCTGCTT GTCAGGCTGTCTGGGatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Beta RNA Construct:</h2><br>p>GTACACCACCTGTGTA AAAGGTTTTGCAC TTTTAATCACTTTTT GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Beta RNA Pathogen-Imitating Region</h2><br><p>AAAAAGTGATTAAAA TTGCAAGAT<br>ACACAGGTGGTGTACatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Gamma RNA Construct:</h2><br><p>GGTTGACAGCTAAAA AAAGGTTTTGCAC GAGCTTTTATTAAATTATAATC GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG<br><br><br><h2>Gamma RNA Pathogen-Imitating Region</h2><br><p>GATTATAATTTAATAAAAGCTC TTAATGATG TTTTAGCTGTCAACCatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Delta RNA Construct:</h2><br><p>TAGGCTAGAGTTGCCAAAGGTTTTGCAC  AAAACCCCCAGACAG GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Delta RNA Pathogen-Imitating Region</h2><br><p>CTGTCTGGGGGTTTTATGGACTGGGGCAACTCTAGCCTAatccttagcgaaagctaaggatttttttt</p><br><br><br><h2>Epsilon RNA Construct:</h2><br><p>TCGAGTTGGATGGCAAAAGGTTTTGCAC  TAATTACACGATTGAATAACCGCC GTGCAAAACCTTTCGCGGTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</p><br><br><br><h2>Epsilon RNA Pathogen-Imitating Region:</h2><br><p>GGCGGTTATTCAATCGTGTAATTATCAATAAAATGCCATCCAACTCGAatccttagcgaaagctaaggatttttttt</p>");