http://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&feed=atom&action=historyTeam:TU Munich/Modeling - Revision history2024-03-28T14:36:55ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208809&oldid=prevSstraub: /* Overview */2010-11-20T16:55:55Z<p><span class="autocomment">Overview</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We simulated the termination and antitermination properties of our transmitter-switch constructs with NUPACK and the kinetic behavior with [http://kinefold.curie.fr/| Kinefold web server] and used some standard estimations for diffusive terms. Our main goal was to prove that our constructs work and that termination is stopped efficiently, that is, that the transmitter molecule binds and antitermination occurs before the RNA polymerase falls off.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We simulated the termination and antitermination properties of our transmitter-switch constructs with NUPACK and the kinetic behavior with [http://kinefold.curie.fr/| Kinefold web server] and used some standard estimations for diffusive terms. Our main goal was to prove that our constructs work and that termination is stopped efficiently, that is, that the transmitter molecule binds and antitermination occurs before the RNA polymerase falls off.<br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">=</del>=Results<del class="diffchange diffchange-inline">=</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Results=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occurred if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynamical approach.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occurred if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynamical approach.</div></td></tr>
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</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208808&oldid=prevSstraub at 16:55, 20 November 20102010-11-20T16:55:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to model a logic gate consisting of two of our switches we introduced above. We proved this logic AND gate to work well, that is if the two input sequences are present the terminators are destroyed and if only one or none signal is present termination occurs.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We were able to model a logic gate consisting of two of our switches we introduced above. We proved this logic AND gate to work well, that is if the two input sequences are present the terminators are destroyed and if only one or none signal is present termination occurs.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">=</del>=Outlook<del class="diffchange diffchange-inline">=</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Outlook=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>So far there is no RNA folding program that is specialized on modeling the interaction of two sequences not to speak of a whole network consisting of RNA based switches and signaling sequences. Combining the concepts of Kinefold<sup>[[Team:TU_Munich/Modeling#ref10|&#91;10&#93;]]</sup> and Vienna<sup>[[Team:TU_Munich/Modeling#ref11|&#91;11&#93;]]</sup> and using sets of secondary structures that are adjusted to having two sequences instead of only one sequence one could get rid of the bulky linkers. The linkers we used did not play a significant role for our simulation as we were just handling small RNA devices, but for larger networks they are likely to be sterically hindering. This obstacle has to be overcome to allow reliable in silcio modeling of RNA devices.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>So far there is no RNA folding program that is specialized on modeling the interaction of two sequences not to speak of a whole network consisting of RNA based switches and signaling sequences. Combining the concepts of Kinefold<sup>[[Team:TU_Munich/Modeling#ref10|&#91;10&#93;]]</sup> and Vienna<sup>[[Team:TU_Munich/Modeling#ref11|&#91;11&#93;]]</sup> and using sets of secondary structures that are adjusted to having two sequences instead of only one sequence one could get rid of the bulky linkers. The linkers we used did not play a significant role for our simulation as we were just handling small RNA devices, but for larger networks they are likely to be sterically hindering. This obstacle has to be overcome to allow reliable in silcio modeling of RNA devices.</div></td></tr>
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</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208807&oldid=prevSstraub at 16:54, 20 November 20102010-11-20T16:54:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our estimations for the switches obtained by the in silico approach indicated that diffusion is negligible. Our RNA dynamics simulations showed for each switch that for appropriate transmitter length the terminator is not folding, hence antitermination is successful. We also found that much smaller transmitter length is suitable for antitermination. Finally, we showed using the example of an AND gate that logic gates based on our transmitter-switch constructs work well.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our estimations for the switches obtained by the in silico approach indicated that diffusion is negligible. Our RNA dynamics simulations showed for each switch that for appropriate transmitter length the terminator is not folding, hence antitermination is successful. We also found that much smaller transmitter length is suitable for antitermination. Finally, we showed using the example of an AND gate that logic gates based on our transmitter-switch constructs work well.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=''In silico'' design <del class="diffchange diffchange-inline">based on thermodynamic calculations</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=''In silico'' design=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=Kinetic <del class="diffchange diffchange-inline">calculations using Kinefold</del>=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Kinetic <ins class="diffchange diffchange-inline">simulations</ins>=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to prove if the antitermination process is not only possible on a thermodynamically base, but also regarding kinetics, we performed the following modeling.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In order to prove if the antitermination process is not only possible on a thermodynamically base, but also regarding kinetics, we performed the following modeling.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208806&oldid=prevSstraub at 16:51, 20 November 20102010-11-20T16:51:36Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our estimations for the switches obtained by the in silico approach indicated that diffusion is negligible. Our RNA dynamics simulations showed for each switch that for appropriate transmitter length the terminator is not folding, hence antitermination is successful. We also found that much smaller transmitter length is suitable for antitermination. Finally, we showed using the example of an AND gate that logic gates based on our transmitter-switch constructs work well.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Our estimations for the switches obtained by the in silico approach indicated that diffusion is negligible. Our RNA dynamics simulations showed for each switch that for appropriate transmitter length the terminator is not folding, hence antitermination is successful. We also found that much smaller transmitter length is suitable for antitermination. Finally, we showed using the example of an AND gate that logic gates based on our transmitter-switch constructs work well.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>=''<del class="diffchange diffchange-inline">in </del>silico'' design based on thermodynamic calculations=</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=''<ins class="diffchange diffchange-inline">In </ins>silico'' design based on thermodynamic calculations=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters. Default 1.0 M Na<sup>+</sup> salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters. Default 1.0 M Na<sup>+</sup> salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==<del class="diffchange diffchange-inline">switches </del>based on attenuation principle==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==<ins class="diffchange diffchange-inline">Switches </ins>based on attenuation principle==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>On a first approach, we designed switches based on terminators performing [[Team:TU_Munich/Glossary#Antitermination| antitermination]] in the context of their natural occurrence within the scope of [[Team:TU_Munich/Glossary#Attenuation| attenuation]], the His and Trp terminator.<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup>. A 20 bp random sequence was derived from [http://www.faculty.ucr.edu/~mmaduro/random.htm| random sequence generator] and put upstream the terminator loop. A complementary sequence, reaching within the terminator´s stem loop (the transmitter) was stepwise shortened to find the length, where the formation of the terminator is thermodynamically favored compared to the strand displacement by this sequence. The final transmitter sequence was then defined by selecting this shortest strand which is still able to "destroy" the stem loop. Subsequently, the complementary random sequence (identity site) was removed an the remaining trigger site was tested in regard of not being able to resolve the stem loop on its on. As illustrated for the His-based switch below, the terminator is thermodynamically favored toward the trigger unit, but in combination with the identity site, destroying the terminator´s stem loop becomes possible. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>On a first approach, we designed switches based on terminators performing [[Team:TU_Munich/Glossary#Antitermination| antitermination]] in the context of their natural occurrence within the scope of [[Team:TU_Munich/Glossary#Attenuation| attenuation]], the His and Trp terminator.<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup>. A 20 bp random sequence was derived from [http://www.faculty.ucr.edu/~mmaduro/random.htm| random sequence generator] and put upstream the terminator loop. A complementary sequence, reaching within the terminator´s stem loop (the transmitter) was stepwise shortened to find the length, where the formation of the terminator is thermodynamically favored compared to the strand displacement by this sequence. The final transmitter sequence was then defined by selecting this shortest strand which is still able to "destroy" the stem loop. Subsequently, the complementary random sequence (identity site) was removed an the remaining trigger site was tested in regard of not being able to resolve the stem loop on its on. As illustrated for the His-based switch below, the terminator is thermodynamically favored toward the trigger unit, but in combination with the identity site, destroying the terminator´s stem loop becomes possible. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==<del class="diffchange diffchange-inline">switches </del>using ubiquitous terminators==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==<ins class="diffchange diffchange-inline">Switches </ins>using ubiquitous terminators==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We applied the same principle to terminators which do not exhibit a natural occurring antitermination and were able to establish switches based on the synthetic terminator BBa_B1006 <sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup> and the T500 terminators<sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup>. We could show the length of the identity site can be varied. Nevertheless, How this will influence kinetic parameters discussed in the [[Team:TU_Munich/Modeling#Kinetic simulations using Kinefold| Kinetic simulations using Kinefold section]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We applied the same principle to terminators which do not exhibit a natural occurring antitermination and were able to establish switches based on the synthetic terminator BBa_B1006 <sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup> and the T500 terminators<sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup>. We could show the length of the identity site can be varied. Nevertheless, How this will influence kinetic parameters discussed in the [[Team:TU_Munich/Modeling#Kinetic simulations using Kinefold| Kinetic simulations using Kinefold section]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''switch based on T500 terminator<sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup>, using a 10 bp recognition site:Comparison of free energy for secondary structure formation at 37°C in the presence and absence of a respective transmitter, simulated by using NUPACK analysis.''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''switch based on T500 terminator<sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup>, using a 10 bp recognition site:Comparison of free energy for secondary structure formation at 37°C in the presence and absence of a respective transmitter, simulated by using NUPACK analysis.''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>==<del class="diffchange diffchange-inline">switches </del>relying on tiny abortive RNAs==</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>==<ins class="diffchange diffchange-inline">Switches </ins>relying on tiny abortive RNAs==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also implemented switches based on a the phenomenon of [[Team:TU_Munich/Glossary#Tiny Abortive RNAs| tiny abortive RNAs]][[Team:TU_Munich/Project#ref5|&#91;5&#93;]]. This principle is in contradiction to NUPACK-simulations, suggesting the T7-terminator to be less stable than simulated in NUPACK. Nevertheless, we tried to implement switches integrating tiny abortive RNAs in different length as trigger site. Results are illustrated in the [[Team:TU_Munich/Project#in vitro Screening| in vitro Screening section.]] </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also implemented switches based on a the phenomenon of [[Team:TU_Munich/Glossary#Tiny Abortive RNAs| tiny abortive RNAs]][[Team:TU_Munich/Project#ref5|&#91;5&#93;]]. This principle is in contradiction to NUPACK-simulations, suggesting the T7-terminator to be less stable than simulated in NUPACK. Nevertheless, we tried to implement switches integrating tiny abortive RNAs in different length as trigger site. Results are illustrated in the [[Team:TU_Munich/Project#in vitro Screening| in vitro Screening section.]] </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxEnd}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxEnd}}</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208805&oldid=prevSstraub: /* in silico design based on thermodynamic calculations */2010-11-20T16:47:23Z<p><span class="autocomment">in silico design based on thermodynamic calculations</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters. Default 1.0 M Na+ salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters. Default 1.0 M Na<ins class="diffchange diffchange-inline"><sup></ins>+<ins class="diffchange diffchange-inline"></sup> </ins>salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==switches based on attenuation principle==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==switches based on attenuation principle==</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208804&oldid=prevSstraub: /* in silico design based on thermodynamic calculations */2010-11-20T16:46:11Z<p><span class="autocomment">in silico design based on thermodynamic calculations</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the following section we will describe how we theoretically designed our switches and their respective transmitter molecules. Our goal was to develop synthetic modified terminators consisting of a target and a recognition site in the described manner (compare [[Team:TU_Munich/Project#Implementation| Implementation]]). As explained, only the complete transmitter RNA may shift the switches´ state, but neither the [[Team:TU_Munich/Glossary#Trigger Site| trigger site]] nor the [[Team:TU_Munich/Glossary#Identity Site| identity site]] alone. This is the prerequisite to fulfill all mentioned requirements for a functional switch allowing to construct logical gates as [[Team:TU_Munich/Project#Implementation| discussed previously]]. We implemented this by optimizing the thermodynamically parameters for pairing of each subsegment [[Team:TU_Munich/Glossary| (identity site, trigger site, recognition site, target site)]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters.Default 1.0 M Na+ salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For all NUPACK simulations, following parameters were used if not mentioned explicitly. A temperature of 37°C was used in combination with the default Serra and Turner (1995) energy parameters. Default 1.0 M Na+ salt concentrations were used. For simulating several strand species, 3 nM switch concentration and 50 nM transmitter concentration were used, which equals conditions in a e.coli cell with a low copy number plasmid coding the switch and accumulation of transmitter RNA.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==switches based on attenuation principle==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==switches based on attenuation principle==</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208803&oldid=prevSstraub: /* Overview */2010-11-20T16:43:46Z<p><span class="autocomment">Overview</span></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We simulated the termination and antitermination properties of our transmitter-switch constructs with <del class="diffchange diffchange-inline">the </del>NUPACK and the <del class="diffchange diffchange-inline">kinetically parameters </del>with [http://kinefold.curie.fr/| Kinefold web server] and used some standard estimations for diffusive terms. Our main goal was to prove that our constructs work and that termination is stopped efficiently, that is, that the transmitter molecule binds and antitermination occurs before the RNA polymerase falls off.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We simulated the termination and antitermination properties of our transmitter-switch constructs with NUPACK and the <ins class="diffchange diffchange-inline">kinetic behavior </ins>with [http://kinefold.curie.fr/| Kinefold web server] and used some standard estimations for diffusive terms. Our main goal was to prove that our constructs work and that termination is stopped efficiently, that is, that the transmitter molecule binds and antitermination occurs before the RNA polymerase falls off.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Results==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Results==</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208802&oldid=prevSstraub: /* References */2010-11-20T16:40:01Z<p><span class="autocomment">References</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref4"></a></html>[4] Larson et al., March 2008, Cell, Volume 132, Issue 6</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref4"></a></html>[4] Larson et al., March 2008, Cell, Volume 132, Issue 6</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref5"></a></html>[5] Lee, S., H.M. Nguyen, and C. Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref5"></a></html>[5] Lee, S., H.M. Nguyen, and C. Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><html><a name="ref6"></a></html>[6] <del class="diffchange diffchange-inline">A. Xayaphoummine</del>, <del class="diffchange diffchange-inline">T</del>. <del class="diffchange diffchange-inline">Bucher and H. Isambert</del>, <del class="diffchange diffchange-inline">Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots, 2005, Nucleic Acids Research, Vol. 33, Web Server issue W605–W610</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><html><a name="ref6"></a></html>[6] <ins class="diffchange diffchange-inline">Berg</ins>, <ins class="diffchange diffchange-inline">v</ins>. <ins class="diffchange diffchange-inline">Hippel</ins>, <ins class="diffchange diffchange-inline">1985</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><html><a name="ref7"></a></html>[7] <del class="diffchange diffchange-inline">Berg</del>, <del class="diffchange diffchange-inline">v</del>. <del class="diffchange diffchange-inline">Hippel, 1985</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><html><a name="ref7"></a></html>[7] <ins class="diffchange diffchange-inline">Doi and Edwards</ins>, <ins class="diffchange diffchange-inline">1999</ins>. <ins class="diffchange diffchange-inline">The Theory of Polymer Dynamics</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><html><a name="ref8"></a></html>[8] <del class="diffchange diffchange-inline">Doi and Edwards</del>, <del class="diffchange diffchange-inline">1999. The Theory of Polymer Dynamics</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><html><a name="ref8"></a></html>[8] <ins class="diffchange diffchange-inline">Rippe</ins>, <ins class="diffchange diffchange-inline">Making contacts on a nucleic acid polymer, 2001, TRENDS in Biochemical Sciences</ins>. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><html><a name="ref9"></a></html>[9] <del class="diffchange diffchange-inline">Rippe</del>, <del class="diffchange diffchange-inline">Making contacts on a nucleic acid polymer</del>, <del class="diffchange diffchange-inline">2001</del>, <del class="diffchange diffchange-inline">TRENDS in Biochemical Sciences</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><html><a name="ref9"></a></html>[9] <ins class="diffchange diffchange-inline">A. Xayaphoummine</ins>, <ins class="diffchange diffchange-inline">T. Bucher and H. Isambert</ins>, <ins class="diffchange diffchange-inline">Kinefold web server for RNA/DNA folding path and structure prediction including pseudoknots and knots</ins>, <ins class="diffchange diffchange-inline">2005, Nucleic Acids Research, Vol. 33, Web Server issue W605–W610</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref10"></a></html>[10] A. Xayaphoummine, T. Bucher, F. Thalmann, and H. Isambert, Prediction and statistics of pseudoknots in RNA structures using exactly clustered stochastic simulations, December 23, 2003, 15310–15315 PNAS vol. 100 no. 26.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref10"></a></html>[10] A. Xayaphoummine, T. Bucher, F. Thalmann, and H. Isambert, Prediction and statistics of pseudoknots in RNA structures using exactly clustered stochastic simulations, December 23, 2003, 15310–15315 PNAS vol. 100 no. 26.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref11"></a></html>[11] Christoph Flamm, Walter Fontana, Ivo L. Hofacker, and Peter Schuster, RNA folding at elementary step resolution, RNA (2000), 6:325–338 Cambridge University Press.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><html><a name="ref11"></a></html>[11] Christoph Flamm, Walter Fontana, Ivo L. Hofacker, and Peter Schuster, RNA folding at elementary step resolution, RNA (2000), 6:325–338 Cambridge University Press.</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208801&oldid=prevSstraub: /* Shifting the switch */2010-11-20T16:38:52Z<p><span class="autocomment">Shifting the switch</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To understand the RNA-folding dynamics of our switches, we performed Kinefold simulations for the His-terminator, the Trp-terminator and for the T7-terminator, each with the corresponding signal. For each terminator-signal construct, there are folding path videos available below and we optimized the signal sequences.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>To understand the RNA-folding dynamics of our switches, we performed Kinefold simulations for the His-terminator, the Trp-terminator and for the T7-terminator, each with the corresponding signal. For each terminator-signal construct, there are folding path videos available below and we optimized the signal sequences.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The Kinefold web server<sup>[[Team:TU_Munich/Modeling#<del class="diffchange diffchange-inline">ref6</del>|&#91;<del class="diffchange diffchange-inline">6</del>&#93;]]</sup> provides a web interface for stochastic folding simulations of nucleic acids and offers the choice of renaturation or co-transcriptional folding. The folding paths are simulated at the level of helix formation and dissociation as these stochastic formation and the removal of individual helices are known to be the limiting steps of RNA folding kinetics.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The Kinefold web server<sup>[[Team:TU_Munich/Modeling#<ins class="diffchange diffchange-inline">ref9</ins>|&#91;<ins class="diffchange diffchange-inline">9</ins>&#93;]]</sup> provides a web interface for stochastic folding simulations of nucleic acids and offers the choice of renaturation or co-transcriptional folding. The folding paths are simulated at the level of helix formation and dissociation as these stochastic formation and the removal of individual helices are known to be the limiting steps of RNA folding kinetics.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For our purposes, co-transcriptional folding was the appropriate choice: Folding proceeds while the sequence is being synthesized from its 5' to 3' ends at a speed of 3 ms per newly added base (for RNA polymerase T7 phage). Thus, the transcript starts to fold before the whole sequence is fully available.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For our purposes, co-transcriptional folding was the appropriate choice: Folding proceeds while the sequence is being synthesized from its 5' to 3' ends at a speed of 3 ms per newly added base (for RNA polymerase T7 phage). Thus, the transcript starts to fold before the whole sequence is fully available.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Kinefold offers the possibility to include additional bases (X) which do not pair to model hybridization dynamics between two sequences.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Kinefold offers the possibility to include additional bases (X) which do not pair to model hybridization dynamics between two sequences.</div></td></tr>
</table>Sstraubhttp://2010.igem.org/wiki/index.php?title=Team:TU_Munich/Modeling&diff=208800&oldid=prevSstraub: /* Diffusion */2010-11-20T16:38:32Z<p><span class="autocomment">Diffusion</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The question whether antitermination occurs is not only guided by the folding process of the signal-terminator pair, but also by how long the signal takes to diffuse to the terminator sequence. The folding of the signal-terminator pair has to be kinetically on the scale of the folding of the terminator structure on its on, to be a valid competitive reaction. This means, the antitermination has to occur before the polymerase will fall of the DNA strand.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The question whether antitermination occurs is not only guided by the folding process of the signal-terminator pair, but also by how long the signal takes to diffuse to the terminator sequence. The folding of the signal-terminator pair has to be kinetically on the scale of the folding of the terminator structure on its on, to be a valid competitive reaction. This means, the antitermination has to occur before the polymerase will fall of the DNA strand.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:TU Munich/Templates/ToggleBoxStart}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To account for the diffusion time, we estimated the hit rate τ<sup>[[Team:TU_Munich/Modeling#<del class="diffchange diffchange-inline">ref7</del>|&#91;<del class="diffchange diffchange-inline">7</del>&#93;]]</sup>, which is the time until the signal meets the terminator sequence for the first time: <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To account for the diffusion time, we estimated the hit rate τ<sup>[[Team:TU_Munich/Modeling#<ins class="diffchange diffchange-inline">ref6</ins>|&#91;<ins class="diffchange diffchange-inline">6</ins>&#93;]]</sup>, which is the time until the signal meets the terminator sequence for the first time: <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:TU_Munich_iGEM2010_sstraub_tau.png |center|100 px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:TU_Munich_iGEM2010_sstraub_tau.png |center|100 px]]</div></td></tr>
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<td colspan="2" class="diff-lineno">Line 214:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>where ''D'' is the diffusion constant, ''a'' the radius of gyration of the signal molecule and ''r'' the radius of the compartment.<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>where ''D'' is the diffusion constant, ''a'' the radius of gyration of the signal molecule and ''r'' the radius of the compartment.<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>For ''E. coli'' ''r'' is approximately 1 μm. The radius of gyration ''a'' can be estimated using the worm-like-chain model<sup>[[Team:TU_Munich/Modeling#<del class="diffchange diffchange-inline">ref8</del>|&#91;<del class="diffchange diffchange-inline">8</del>&#98;]]</sup> by <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For ''E. coli'' ''r'' is approximately 1 μm. The radius of gyration ''a'' can be estimated using the worm-like-chain model<sup>[[Team:TU_Munich/Modeling#<ins class="diffchange diffchange-inline">ref7</ins>|&#91;<ins class="diffchange diffchange-inline">7</ins>&#98;]]</sup> by <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:TU_Munich_iGEM2010_sstraub_a.png |center|100 px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:TU_Munich_iGEM2010_sstraub_a.png |center|100 px]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>where ''n'' is the length of the signal which is 0,3 nm/monomer, ''l'' is the persistency length<sup>[[Team:TU_Munich/Modeling#<del class="diffchange diffchange-inline">ref9</del>|&#91;<del class="diffchange diffchange-inline">9</del>&#93;]]</sup> which is 2nm for single-stranded RNA. Thus, for a signal of length 32 nt, ''a'' = 6,4 nm.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>where ''n'' is the length of the signal which is 0,3 nm/monomer, ''l'' is the persistency length<sup>[[Team:TU_Munich/Modeling#<ins class="diffchange diffchange-inline">ref8</ins>|&#91;<ins class="diffchange diffchange-inline">8</ins>&#93;]]</sup> which is 2nm for single-stranded RNA. Thus, for a signal of length 32 nt, ''a'' = 6,4 nm.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The diffusion constant ''D'' was obtained by <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The diffusion constant ''D'' was obtained by <br></div></td></tr>
</table>Sstraub