http://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&feed=atom&action=historyRice/Project A: Phage Project - Revision history2024-03-28T15:20:14ZRevision history for this page on the wikiMediaWiki 1.16.5http://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47959&oldid=prevBibhash: /* '''Results''' */2007-10-27T03:57:13Z<p><span class="autocomment">'''Results'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The probability of lysis given a specific induction of the circuit by tetracycline. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The probability of lysis given a specific induction of the circuit by tetracycline. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The modeling exercise provided us a proof of principle that it should be possible to engineer production of lambda repressor under the control of tetracycline such that tetracycline resistant cells are under a fitness disadvantage regime and are out competed by the tetracycline sensitive cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The modeling exercise provided us a proof of principle that it should be possible to engineer production of lambda repressor under the control of tetracycline such that tetracycline resistant cells are under a fitness disadvantage regime and are out competed by the tetracycline sensitive cells <ins class="diffchange diffchange-inline">(Figure I)</ins>. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Before, the full scale simulation was run, preliminary models were run in order to better understand the results of the different plasmid constructs. These experimental results helped to fine tune the parameters of the model. This refined model could then predict the dynamic of the engineered circuit within the phage genome in order to understand the effects of stochasticity and gene copy number on the efficacy of the circuit's ability to decrease the fitness of resistant cells. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Before, the full scale simulation was run, preliminary models were run in order to better understand the results of the different plasmid constructs. These experimental results helped to fine tune the parameters of the model. This refined model could then predict the dynamic of the engineered circuit within the phage genome in order to understand the effects of stochasticity and gene copy number on the efficacy of the circuit's ability to decrease the fitness of resistant cells <ins class="diffchange diffchange-inline">(Figure J)</ins>. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the early stages of designed the circuit, the negative feedback on TetR expression was added in order to decease the noise in the circuit and to provide more stable expression levels of TetR for varying gene copy numbers as predicted by the preliminary model. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In the early stages of designed the circuit, the negative feedback on TetR expression was added in order to decease the noise in the circuit and to provide more stable expression levels of TetR for varying gene copy numbers as predicted by the preliminary model. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Tableoparts.jpg|thumb|right|500px| Cells were cultured in LB. +RQEλ55 and +B0011 cells were cultured with 50ug/mL ampicillin. Doubling time was found by measuring optical density (OD600nm) of cultures at hour intervals for five hours.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Tableoparts.jpg|thumb|right|500px| <ins class="diffchange diffchange-inline">K. </ins>Cells were cultured in LB. +RQEλ55 and +B0011 cells were cultured with 50ug/mL ampicillin. Doubling time was found by measuring optical density (OD600nm) of cultures at hour intervals for five hours.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The doubling times listed in the table to the left provide the relative fitness of TetS and TetR cells grown at different temperatures containing different circuits. The data indicate that, at 37 degrees C, our circuit does not impact the doubling time of TetR or TetS cells. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The doubling times listed in the table to the left provide the relative fitness of TetS and TetR cells grown at different temperatures containing different circuits. The data indicate that, at 37 degrees C, our circuit does not impact the doubling time of TetR or TetS cells. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:FluorescentData.jpg|thumb|right|500px| Top: Averaged fluorescent data of taken from four M9 minimal media cultures of TetS+RQEλ55 and TetS+B0011 over the course of 90 minutes. The excitation and emission wavelengths used to measure fluorescence were 514nm and 527nm respectively. Bottom:Averaged fluorescent data of taken from four M9 minimal media cultures of TetR+RQEλ55 and TetR+B0011 over the course of 90 minutes. The excitation and emission wavelengths used to measure fluorescence were 514nm and 527nm respectively.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:FluorescentData.jpg|thumb|right|500px|<ins class="diffchange diffchange-inline">L. </ins>Top: Averaged fluorescent data of taken from four M9 minimal media cultures of TetS+RQEλ55 and TetS+B0011 over the course of 90 minutes. The excitation and emission wavelengths used to measure fluorescence were 514nm and 527nm respectively. Bottom:Averaged fluorescent data of taken from four M9 minimal media cultures of TetR+RQEλ55 and TetR+B0011 over the course of 90 minutes. The excitation and emission wavelengths used to measure fluorescence were 514nm and 527nm respectively.]]</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">The following figure </del>measures the relative fluorescence of TetR and TetS cells with and without our circuit. The cells were cultured at 37 degrees C in M9 minimal media with 50ug/mL ampicillin and 1uM ATc. 1uM ATc is assumed to fully induce our circuit and produce a bolus of EYFP and CI in both TetS and TetR cells. The effectiveness of our circuit will be determined by the ratio of the fluorescence produced by TetS TetR. A high TetS/TetR ratio indicates that TetS cells are producing much more CI than the TetR cells, making any TetS lysogen much less likely to go into lytic phase. The data show that the fluorescence levels of TetR+RQEλ55 + are negligible when compared to the autofluorescence of TetR+B0011 while TetS+RQEλ55 produces a noticeable amount of fluorescence when compared to the TetS+B0011. At 100% induction, it appears that our circuit it binary. TetS+RQEλ55 cells will produce EYFP and CI while TetR+RQEλ55 produce almost no EYFP and CI.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Figure K and L </ins>measures the relative fluorescence of TetR and TetS cells with and without our circuit. The cells were cultured at 37 degrees C in M9 minimal media with 50ug/mL ampicillin and 1uM ATc. 1uM ATc is assumed to fully induce our circuit and produce a bolus of EYFP and CI in both TetS and TetR cells. The effectiveness of our circuit will be determined by the ratio of the fluorescence produced by TetS TetR. A high TetS/TetR ratio indicates that TetS cells are producing much more CI than the TetR cells, making any TetS lysogen much less likely to go into lytic phase. The data show that the fluorescence levels of TetR+RQEλ55 + are negligible when compared to the autofluorescence of TetR+B0011 while TetS+RQEλ55 produces a noticeable amount of fluorescence when compared to the TetS+B0011. At 100% induction, it appears that our circuit it binary. TetS+RQEλ55 cells will produce EYFP and CI while TetR+RQEλ55 produce almost no EYFP and CI.</div></td></tr>
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</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47935&oldid=prevBibhash: /* '''Predictive Modeling of the Circuit''' */2007-10-27T03:54:54Z<p><span class="autocomment">'''Predictive Modeling of the Circuit'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Later, we used the model to analyze the effects of altering the strength of cI repression by TetR as well as the negative feedback of TetR.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Later, we used the model to analyze the effects of altering the strength of cI repression by TetR as well as the negative feedback of TetR.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|400px|right|Schematic of modeling output]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|400px|right|<ins class="diffchange diffchange-inline">I. </ins>Schematic of modeling output]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HighPlasmidMeanEyfp.jpg.jpg|thumb|'''HighPlasmidMeanEyfp'''|400px|right|Difference between Single Cell Fluorescence levels in Tet sensitive versus resistant cells. The example showin in the model has a plasmid copy number of 100 per bacterial cell (of the circuit shown in Figure C and the readout is EYFP fluorescence]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HighPlasmidMeanEyfp.jpg.jpg|thumb|'''HighPlasmidMeanEyfp'''|400px|right|<ins class="diffchange diffchange-inline">J. </ins>Difference between Single Cell Fluorescence levels in Tet sensitive versus resistant cells. The example showin in the model has a plasmid copy number of 100 per bacterial cell (of the circuit shown in Figure C and the readout is EYFP fluorescence]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Characterization of the Circuit''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Characterization of the Circuit''' ====</div></td></tr>
</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47906&oldid=prevTstevenson: /* '''Experimental System and Circuit Design''' */2007-10-27T03:51:57Z<p><span class="autocomment">'''Experimental System and Circuit Design'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In order to set the stage for playing evolutionary competition games, we first engineer a bacterial population consisting of ''E. coli'' cells that are either sensitive or resistant to the application of the antibiotic tetracycline. Our goal is to make tetracycline <del class="diffchange diffchange-inline">sensitivite </del>cells more fit than tetracycline resistant cells. The ability to survive increasing concentrations of tetracycline is used as a measure of fitness. This idea is summarized in the Figures A and B: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to set the stage for playing evolutionary competition games, we first engineer a bacterial population consisting of ''E. coli'' cells that are either sensitive or resistant to the application of the antibiotic tetracycline. Our goal is to make tetracycline <ins class="diffchange diffchange-inline">sensitive </ins>cells more fit than tetracycline resistant cells. The ability to survive increasing concentrations of tetracycline is used as a measure of fitness. This idea is summarized in the Figures A and B: </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:circuit_overview.jpg|thumb|500px|right|'''C.''' Modular parts of the circuit to be constructed.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:circuit_overview.jpg|thumb|500px|right|'''C.''' Modular parts of the circuit to be constructed.]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:full_circuit_biobrick.jpg|thumb|600px|center|'''G.'''The complete Biobricks circuit.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:full_circuit_biobrick.jpg|thumb|600px|center|'''G.'''The complete Biobricks circuit.]]</div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">==== '''Constructs Submitted and Verified''' ====</ins></div></td></tr>
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</table>Tstevensonhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47877&oldid=prevBibhash: /* '''Predictive Modeling of the Circuit''' */2007-10-27T03:49:43Z<p><span class="autocomment">'''Predictive Modeling of the Circuit'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Predictive Modeling of the Circuit''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Predictive Modeling of the Circuit''' ====</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The modeling <del class="diffchange diffchange-inline">expercise </del>provided us a proof <del class="diffchange diffchange-inline">the </del>principle that it should be possible to engineer production of lambda repressor under the control of Tetracycline such that Tetracycline resistant cells are under a fitness disadvantage regime and are out competed by the Tetracycline sensitive cells. From this model, we intend to extract the follwing information: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The modeling <ins class="diffchange diffchange-inline">exercise </ins>provided us <ins class="diffchange diffchange-inline">with </ins>a proof<ins class="diffchange diffchange-inline">-of-</ins>principle that it should be possible to engineer production of lambda repressor under the control of Tetracycline such that Tetracycline resistant cells are under a fitness disadvantage regime and are out competed by the Tetracycline sensitive cells. From this model, we intend to extract the follwing information: </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Concentration profile of Tetracycline necessary for induction of the circuit. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Concentration profile of Tetracycline necessary for induction of the circuit. </div></td></tr>
</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47839&oldid=prevTstevenson: /* '''Modeling''' */2007-10-27T03:45:31Z<p><span class="autocomment">'''Modeling'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We use stochastic differential equations to represent chemical reactions with each reacting species being a component of the circuit. Numerical solutions are obtained by the '''[[Gillespie Stochastic Simulation Algorithm]]''', implemented in Matlab. Here is the [[code]] used. Figure H shows the reactions used in the modeling. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We use stochastic differential equations to represent chemical reactions with each reacting species being a component of the circuit. Numerical solutions are obtained by the '''[[Gillespie Stochastic Simulation Algorithm]]''', implemented in Matlab. Here is the [[code]] used. Figure H shows the reactions used in the modeling. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The Gillespie algorithm is used to perform a discrete stochastic simulation of a system of well mixed reactant molecules wherein the 'fate trajectory' of each molecule is explicitly simulated. A Gillespie simulation is a random walk that exactly represents the distribution of the Chemical Master Equation. The physical interpretation of the Simulation is that individual molecules participating as reactants in a system undergo frequent collisions, a sub-set of which are collisions that are in correct stereo-chemical orientation and with sufficient potential energy. In a Gillespie Simulation exercise, a well mixed reaction environment is presumed. Each of the participating species <del class="diffchange diffchange-inline">are </del>explicitly traced <del class="diffchange diffchange-inline">such </del>that (a) which reaction will occur at the next time step and (b) probability of a reaction occurring at the next time step<del class="diffchange diffchange-inline">, </del>is known. The Gillespie method of stochastic simulation is advantageous over the deterministic differential equation based modeling of chemical reactions assuming mass action kinetics because <del class="diffchange diffchange-inline">this </del>does not approximate chemical reactions as occurring in bulk<del class="diffchange diffchange-inline">, </del>rather accounts for a chemical reaction occurring between two chemical species. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The Gillespie algorithm is used to perform a discrete stochastic simulation of a system of well mixed reactant molecules wherein the 'fate trajectory' of each molecule is explicitly simulated. A Gillespie simulation is a random walk that exactly represents the distribution of the Chemical Master Equation. The physical interpretation of the Simulation is that individual molecules participating as reactants in a system undergo frequent collisions, a sub-set of which are collisions that are in correct stereo-chemical orientation and with sufficient potential energy. In a Gillespie Simulation exercise, a well mixed reaction environment is presumed. Each of the participating species <ins class="diffchange diffchange-inline">is </ins>explicitly traced <ins class="diffchange diffchange-inline">so </ins>that (a) which reaction will occur at the next time step and (b) <ins class="diffchange diffchange-inline">the </ins>probability of a reaction occurring at the next time step is known. The Gillespie method of stochastic simulation is advantageous over the deterministic differential equation based modeling of chemical reactions assuming mass action kinetics because <ins class="diffchange diffchange-inline">it </ins>does not approximate chemical reactions as occurring in bulk <ins class="diffchange diffchange-inline">but </ins>rather accounts for a chemical reaction occurring between two chemical species. </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:Model_SSAequations.jpg|thumb|400px|right|'''H.''' Chemical Equations used in the Gillespie SSA solution]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Model_SSAequations.jpg|thumb|400px|right|'''H.''' Chemical Equations used in the Gillespie SSA solution]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The following '''assumptions''' are made: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The following '''assumptions''' are made: </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>* Any spatial aspects of the modeling problem <del class="diffchange diffchange-inline">is </del>ignored (i.e. Gillespie is only running in time)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Any spatial aspects of the modeling problem <ins class="diffchange diffchange-inline">are </ins>ignored (i.e. Gillespie is only running in time)</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>* Parameter values are input from the following references:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Parameter values are input from the following references:</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Sotiropoulos, V. and Kaznessis, Y. N. Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes. BMC Systems Biology. Jan 2009.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Sotiropoulos, V. and Kaznessis, Y. N. Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes. BMC Systems Biology. Jan 2009.</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>* Steps in gene expression i.e. transcription and translation are simplified by not modeling RNAP binding/unbinding to the promoter and Ribsome binding/unbinding; modified propensities are used from the reference above.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Steps in gene expression <ins class="diffchange diffchange-inline">(</ins>i.e. transcription and translation<ins class="diffchange diffchange-inline">) </ins>are simplified by not modeling RNAP binding/unbinding to the promoter and Ribsome binding/unbinding; modified propensities are used from the reference above.</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>* Growth rates are assumed not to vary among cell types (i.e. infected vs. non-infected).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Growth rates are assumed not to vary among cell types (i.e. infected vs. non-infected).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The propensities for tetA is recovered from experimental data in the literature.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The propensities for tetA is recovered from experimental data in the literature.</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>For the circuit constructed, one of the possible readouts of 'measure of fitness' in the wake of <del class="diffchange diffchange-inline">Tetracycline </del>exposure would be the amount of CI produced. Through the simulations, we trace the time evolution profiles of CI production and compare the results in Tet-sensitive and Tet-resistant cells. We infer the probability of a population of Tet-resistant ''E.coli'' cells going into lysis over the choice to remain in lysogenic state increases after <del class="diffchange diffchange-inline">Tetracycline </del>challenge. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>For the circuit constructed, one of the possible readouts of 'measure of fitness' in the wake of <ins class="diffchange diffchange-inline">tetracycline </ins>exposure would be the amount of CI produced. Through the simulations, we trace the time evolution profiles of CI production and compare the results in Tet-sensitive and Tet-resistant cells. We infer <ins class="diffchange diffchange-inline">that </ins>the probability of a population of Tet-resistant ''E. coli'' cells going into lysis over the choice to remain in lysogenic state increases after <ins class="diffchange diffchange-inline">tetracycline </ins>challenge. </div></td></tr>
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<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>
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</table>Tstevensonhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47832&oldid=prevBibhash: /* '''Predictive Modeling of the Circuit''' */2007-10-27T03:45:09Z<p><span class="autocomment">'''Predictive Modeling of the Circuit'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|400px|right|Schematic of modeling output]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|400px|right|Schematic of modeling output]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HighPlasmidMeanEyfp.jpg.jpg|thumb|'''HighPlasmidMeanEyfp'''|400px|right|Difference between Single Cell Fluorescence levels in Tet sensitive versus resistant cells ]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HighPlasmidMeanEyfp.jpg.jpg|thumb|'''HighPlasmidMeanEyfp'''|400px|right|Difference between Single Cell Fluorescence levels in Tet sensitive versus resistant cells<ins class="diffchange diffchange-inline">. The example showin in the model has a plasmid copy number of 100 per bacterial cell (of the circuit shown in Figure C and the readout is EYFP fluorescence</ins>]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Characterization of the Circuit''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Characterization of the Circuit''' ====</div></td></tr>
</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47811&oldid=prevBibhash: /* '''Milestones Accomplished''' */2007-10-27T03:42:37Z<p><span class="autocomment">'''Milestones Accomplished'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Milestones Accomplished''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Milestones Accomplished''' ==</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Watch out for our presentation and poster at the Jamboree!</ins></div></td></tr>
</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47806&oldid=prevTstevenson: /* '''Experimental System and Circuit Design''' */2007-10-27T03:42:04Z<p><span class="autocomment">'''Experimental System and Circuit Design'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</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 order to set the stage for playing evolutionary competition games, we first engineer a bacterial population consisting of ''E.coli'' cells that are either sensitive or resistant to the application of the antibiotic tetracycline. Our goal is to make <del class="diffchange diffchange-inline">cells harboring </del>tetracycline <del class="diffchange diffchange-inline">sensitivity </del>more fit than <del class="diffchange diffchange-inline">cells with </del>tetracycline <del class="diffchange diffchange-inline">resistance</del>. <del class="diffchange diffchange-inline">Ability </del>to survive increasing <del class="diffchange diffchange-inline">concentration </del>of tetracycline is used as a measure of fitness. <del class="diffchange diffchange-inline">The </del>idea is summarized in the <del class="diffchange diffchange-inline">figures </del>A and B: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In order to set the stage for playing evolutionary competition games, we first engineer a bacterial population consisting of ''E. coli'' cells that are either sensitive or resistant to the application of the antibiotic tetracycline. Our goal is to make tetracycline <ins class="diffchange diffchange-inline">sensitivite cells </ins>more fit than tetracycline <ins class="diffchange diffchange-inline">resistant cells</ins>. <ins class="diffchange diffchange-inline">The ability </ins>to survive increasing <ins class="diffchange diffchange-inline">concentrations </ins>of tetracycline is used as a measure of fitness. <ins class="diffchange diffchange-inline">This </ins>idea is summarized in the <ins class="diffchange diffchange-inline">Figures </ins>A and B: </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:circuit_overview.jpg|thumb|500px|right|'''C.''' Modular parts of the circuit to be constructed.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:circuit_overview.jpg|thumb|500px|right|'''C.''' Modular parts of the circuit to be constructed.]]</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>'''MIC''': <del class="diffchange diffchange-inline">the </del>lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in laboratory culture conditions to confirm resistance of microorganisms to an antimicrobial agent and to monitor the activity of new antimicrobial agents.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''MIC''': <ins class="diffchange diffchange-inline">The </ins>lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in laboratory culture conditions to confirm resistance of microorganisms to an antimicrobial agent and to monitor the activity of new antimicrobial agents.</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>A bacterial cell infected with <del class="diffchange diffchange-inline">Bacteriophage </del>lambda regulates the lysis versus lysogeny choice by modulating the amount of lambda repressor produced. High lambda repressor downregulates phage genes concurrently <del class="diffchange diffchange-inline">upregulating </del>genes that allow it to remain in lysogeny. However, the lysis pathway switch is flipped upon decrease of lambda repressor brought about by induction by exposure to UV <del class="diffchange diffchange-inline">causing </del>increase Cro protein. It follows that increased immunity to lambda can be induced by increasing concentration of lambda repressor, allowing the bacteria to remain in lysogeny. We simultaneously couple the lytic-lysogeny switch to tetracycline induced transcription of lambda repressor <del class="diffchange diffchange-inline">within one bacterial cell </del>as well as tetracycline induced antibiotic resistance to comparison of fitness between resistant and sensitive cells<del class="diffchange diffchange-inline">, in order to characterize the proof of principle of selection against resistance</del>. By this coupling, we want to tune the parameters in our system <del class="diffchange diffchange-inline">such </del>that we land in <del class="diffchange diffchange-inline">the </del>fitness regime that will confer selective advantage to the cells <del class="diffchange diffchange-inline">with Tetracycline sensitivity</del>, simultaneously <del class="diffchange diffchange-inline">lowering the lethality of </del>cells <del class="diffchange diffchange-inline">with Tetracycline resistance</del>. <del class="diffchange diffchange-inline">The </del>idea is summarized in <del class="diffchange diffchange-inline">the figure below</del>: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>A bacterial cell infected with <ins class="diffchange diffchange-inline">bacteriophage </ins>lambda regulates the lysis versus lysogeny choice by modulating the amount of lambda repressor produced. High lambda repressor downregulates phage genes <ins class="diffchange diffchange-inline">and </ins>concurrently <ins class="diffchange diffchange-inline">upregulated its own </ins>genes that allow it to remain in lysogeny. However, the lysis pathway switch is flipped <ins class="diffchange diffchange-inline">on </ins>upon decrease of lambda repressor<ins class="diffchange diffchange-inline">. This is </ins>brought about by induction by exposure to UV<ins class="diffchange diffchange-inline">, which causes an </ins>increase <ins class="diffchange diffchange-inline">of </ins>Cro protein. It follows that increased immunity to lambda can be induced by increasing concentration of <ins class="diffchange diffchange-inline">the </ins>lambda repressor, allowing the bacteria to remain in lysogeny. We simultaneously couple the lytic-lysogeny switch to tetracycline induced transcription of lambda repressor as well as tetracycline induced antibiotic resistance to comparison of fitness between resistant and sensitive cells. By this coupling, we want to tune the parameters in our system <ins class="diffchange diffchange-inline">so </ins>that we land in <ins class="diffchange diffchange-inline">a </ins>fitness regime that will confer selective advantage to the <ins class="diffchange diffchange-inline">tetracycline sensitive </ins>cells, simultaneously <ins class="diffchange diffchange-inline">selecting againt tetracycline resistant </ins>cells. <ins class="diffchange diffchange-inline">This </ins>idea is summarized in <ins class="diffchange diffchange-inline">Figure D</ins>: </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:tet_sensor.jpg|thumb|500px|right|'''D.''' Output of the circuit to presence or absence of tetracycline.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:tet_sensor.jpg|thumb|500px|right|'''D.''' Output of the circuit to presence or absence of tetracycline.]]</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>'''Basic Circuit Design''' - The general strategy for linking lysis to tetracycline resistance<del class="diffchange diffchange-inline">, </del>involves putting a lambda transcription factor (CI) under control of the Tet promoter. <del class="diffchange diffchange-inline"> </del>This circuit <del class="diffchange diffchange-inline">makes </del>the amount of CI in a cell proportional to the amount of tetracycline in the cell. <del class="diffchange diffchange-inline"> </del>Cells with higher concentrations of tetracycline have higher levels of CI thus preventing lysis of the cell via phage. <del class="diffchange diffchange-inline"> </del>Cells with lower levels of tetracycline (cells that are resistant to tetracycline) will have less CI present and thus be more <del class="diffchange diffchange-inline">likely </del>to <del class="diffchange diffchange-inline">be lysed </del>by phage. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Basic Circuit Design''' - The general strategy for linking lysis to tetracycline resistance involves putting a lambda transcription factor (CI) under control of the Tet promoter. This circuit <ins class="diffchange diffchange-inline">causes </ins>the amount of CI in a cell <ins class="diffchange diffchange-inline">to be </ins>proportional to the amount of tetracycline in the cell. Cells with higher concentrations of tetracycline have higher levels of CI<ins class="diffchange diffchange-inline">, </ins>thus preventing lysis of the cell via phage. Cells with lower levels of tetracycline (cells that are resistant to tetracycline) will have less CI present and thus be more <ins class="diffchange diffchange-inline">susceptible </ins>to <ins class="diffchange diffchange-inline">lysis </ins>by phage. </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:tc sensor function.jpg|thumb|500px|right|'''E.''' Biobrick parts used in construction of the Tet sensor.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:tc sensor function.jpg|thumb|500px|right|'''E.''' Biobrick parts used in construction of the Tet sensor.]]</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>We designed the circuit keeping in mind the following <del class="diffchange diffchange-inline">attributes</del>: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We designed the circuit keeping in mind the following <ins class="diffchange diffchange-inline">goals</ins>: </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">a </del>circuit with existing BioBricks requiring few ligations to construct </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <ins class="diffchange diffchange-inline">A </ins>circuit with existing BioBricks requiring few ligations to construct </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">a </del>design that could be used with multiple types of resistance</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <ins class="diffchange diffchange-inline">A </ins>design that could be used with multiple types of resistance</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">a </del>robust circuit that will remain stable for large numbers of generations</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <ins class="diffchange diffchange-inline">A </ins>robust circuit that will remain stable for large numbers of generations</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">a </del>circuit that can propagate through bacterial populations, e.g., practical for treating environmental problems</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <ins class="diffchange diffchange-inline">A </ins>circuit that can propagate through bacterial populations, e.g., practical for treating environmental problems<ins class="diffchange diffchange-inline">.</ins></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>'''Antibiotic Selection''' - The antibiotic resistance we will be selecting against in this circuit will be tetracycline. <del class="diffchange diffchange-inline"> </del>Tetracycline was selected because Tn10 tetracycline resistance transposon has been extremely well characterized and can be activated by a non antibiotic molecule anhydrotetracycline that binds to the TetR transcription factor, allowing us to use a variety of approaches when characterizing the genetic circuit. TetA, the tetracycline efflux pump coded for by Tn10, is toxic to cells. <del class="diffchange diffchange-inline"> </del>As a result, under conditions of low or no tetracyline, the tetracycline sensitive will already have a slight advantage over the tetracycline resistant cells which will reduce the amount of tuning required for this circuit. <del class="diffchange diffchange-inline"> Also</del>, the human body is extremely tolerant of tetracycline, making it a good target for resistance reduction.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Antibiotic Selection''' - The antibiotic resistance we will be selecting against in this circuit will be tetracycline. Tetracycline was selected because <ins class="diffchange diffchange-inline">the </ins>Tn10 tetracycline resistance transposon has been extremely well characterized and can be activated by a non<ins class="diffchange diffchange-inline">-</ins>antibiotic molecule anhydrotetracycline <ins class="diffchange diffchange-inline">(ATc) </ins>that binds to the TetR transcription factor, allowing us to use a variety of approaches when characterizing the genetic circuit. TetA, the tetracycline efflux pump coded for by Tn10, is toxic to cells. As a result, under conditions of low or no tetracyline, the tetracycline sensitive <ins class="diffchange diffchange-inline">cells </ins>will already have a slight advantage over the tetracycline resistant cells<ins class="diffchange diffchange-inline">, </ins>which will reduce the amount of tuning required for this circuit. <ins class="diffchange diffchange-inline">Furthermore</ins>, the human body is extremely tolerant of tetracycline, making it a good target for resistance reduction.</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>[[Image:Lambda CI generator.jpg|thumb|250px|right|'''F.''' <del class="diffchange diffchange-inline">Biobricks </del>parts used in construction of the Lambda <del class="diffchange diffchange-inline">generator</del>.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Lambda CI generator.jpg|thumb|250px|right|'''F.''' <ins class="diffchange diffchange-inline">Biobrick </ins>parts used in construction of the Lambda <ins class="diffchange diffchange-inline">Generator</ins>.]]</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>'''Strain Selection''' – To compare the relative fitness of tetracycline resistant versus that of tetracycline sensitive phage infected cells, we selected two strains of ''E.coli'' genotypically identical with the exception that one has the Tn10 tetracyline resistance transposon (<del class="diffchange diffchange-inline">GNB8385K,tetracyline sensitive & GNB824,tetracyline </del>resistant)<del class="diffchange diffchange-inline">.GNB824 </del>is a derivative GNB8385K that had the Tn10 transposon incorporated genomically. <del class="diffchange diffchange-inline"> </del>Tn10 incorporation was screened for by assaying loss of regulation of the lysine decarboxylase loci, resulting in pH independent production of lysine decarboxylase (cells with properly incorporated transposon turn red on MacConkey agar). <del class="diffchange diffchange-inline"> </del>These nearly identical strains will allow us to <del class="diffchange diffchange-inline">asses </del>the impact of tetracycline resistance on relative fitness. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Strain Selection''' – To compare the relative fitness of tetracycline resistant <ins class="diffchange diffchange-inline">cells </ins>versus that of tetracycline sensitive phage infected cells, we selected two strains of ''E. coli'' genotypically identical with the exception that one has the Tn10 tetracyline resistance transposon<ins class="diffchange diffchange-inline">. GNB824 </ins>(<ins class="diffchange diffchange-inline">tetracycline </ins>resistant) is a derivative GNB8385K <ins class="diffchange diffchange-inline">(tetracycline sensitive) </ins>that had the Tn10 transposon incorporated genomically. Tn10 incorporation was screened for by assaying loss of regulation of the lysine decarboxylase loci, resulting in pH independent production of lysine decarboxylase (cells with properly incorporated transposon turn red on MacConkey agar). These nearly identical strains will allow us to <ins class="diffchange diffchange-inline">assess </ins>the impact of tetracycline resistance on relative fitness. </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>'''Bacteriophage Selection''' - When selecting the bacteriophage to be used in this circuit, we desired a phage whose life cycle and genome had been well characterized. Bacteriophage lambda has all of <del class="diffchange diffchange-inline">these </del>characteristics as well as the benefit of being commonly used as a cloning vector. Specifically, the lambda strain we selected can be induced to undergo lytic growth by growing lysogenic cells at 42°C. Also, due to the infectious nature of bacteriophage lambda, we selected a strain that is unable to lyse cells (amber mutation <del class="diffchange diffchange-inline">preventing </del>the expression of lytic enzyme, protecting other bacteria within the lab from phage infection). <del class="diffchange diffchange-inline"> </del>Though the phage will not lyse the cells, it will produce fully functional phage particles that can be isolated via chloroform extraction. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Bacteriophage Selection''' - When selecting the bacteriophage to be used in this circuit, we desired a phage whose life cycle and genome had been well characterized. Bacteriophage lambda has all of <ins class="diffchange diffchange-inline">this </ins>characteristics as well as the benefit of being commonly used as a cloning vector. Specifically, the lambda strain we selected can be induced to undergo lytic growth by growing lysogenic cells at 42°C. Also, due to the infectious nature of bacteriophage lambda, we selected a strain that is unable to lyse cells (amber mutation <ins class="diffchange diffchange-inline">prevents </ins>the expression of lytic enzyme, protecting other bacteria within the lab from phage infection). Though the phage will not lyse the cells, it will produce fully functional phage particles that can be isolated via chloroform extraction. </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>'''Phage Engineering''' – We will construct a recombination plasmid designed to integrate our circuit into lambda DNA located chromosomally in lysogen ''E.coli''. The plasmid will not contain an origin of replication (to limit recombination sites), so we will have to amplify it using high fidelity PCR. <del class="diffchange diffchange-inline"> </del>The plasmid will contain our circuit flanked by homologous recombination sites that will combine at <del class="diffchange diffchange-inline"> </del>non-critical location of the phage chromosome (yet to be determined). <del class="diffchange diffchange-inline"> </del>Within this homologous recombination site, we will construct a BioBrick restriction site, allowing us to integrate any BioBrick part into the lambda phage genome. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Phage Engineering''' – We will construct a recombination plasmid designed to integrate our circuit into lambda DNA located chromosomally in lysogen ''E. coli''. The plasmid will not contain an origin of replication (to limit recombination sites), so we will have to amplify it using high fidelity PCR. The plasmid will contain our circuit flanked by homologous recombination sites that will combine at <ins class="diffchange diffchange-inline">a </ins>non-critical location of the phage chromosome (yet to be determined). Within this homologous recombination site, we will construct a BioBrick restriction site, allowing us to integrate any BioBrick part into the lambda phage genome. </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>'''Circuit Characterization''' – To assay relative fitness of the cells, we will measure doubling times of the strains at sub-MIC <del class="diffchange diffchange-inline">(minimum inhibitory concentration) </del>tetracycline by measuring optical density (<del class="diffchange diffchange-inline">OD at </del>600nm) of cells.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>'''Circuit Characterization''' – To assay relative fitness of the cells, we will measure doubling times of the strains at sub-MIC tetracycline by measuring optical density (600nm) of cells.</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>==== '''Experimental Materials and Methods ''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Experimental Materials and Methods ''' ====</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 overall '''construction scheme''' with the modular parts of the circuit is <del class="diffchange diffchange-inline">mentioned </del>in Figure C. Differential response to exposure to <del class="diffchange diffchange-inline">Tetracycline </del>is schematized in Figure D. Biobrick parts used in the circuit <del class="diffchange diffchange-inline">is mentioned </del>in Figure E. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The overall '''construction scheme''' with the modular parts of the circuit is <ins class="diffchange diffchange-inline">shown </ins>in Figure C. Differential response to exposure to <ins class="diffchange diffchange-inline">tetracycline </ins>is schematized in Figure D. Biobrick parts used in the circuit <ins class="diffchange diffchange-inline">are shown </ins>in Figure E. </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>==== '''Testing and Characterization of the Constructed Circuit''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Testing and Characterization of the Constructed Circuit''' ====</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>Using promoters of different strengths, the circuit has been characterized for its ability to grow in various concentrations of <del class="diffchange diffchange-inline">Tetracycline</del>. The EYFP fluorescent reporter serves as a readout for the functionality of the circuit as a Tet sensor. <del class="diffchange diffchange-inline"> </del>The Lambda Generator part is described in Figure F. The final circuit is presented below, in Figure G.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Using promoters of different strengths, the circuit has been characterized for its ability to grow in various concentrations of <ins class="diffchange diffchange-inline">tetracycline</ins>. The EYFP fluorescent reporter serves as a readout for the functionality of the circuit as a Tet sensor. The Lambda Generator part is described in Figure F. The final circuit is presented below, in Figure G.</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:full_circuit_biobrick.jpg|thumb|600px|center|'''G.'''The complete Biobricks circuit.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:full_circuit_biobrick.jpg|thumb|600px|center|'''G.'''The complete Biobricks circuit.]]</div></td></tr>
</table>Tstevensonhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47737&oldid=prevBibhash: /* '''Results''' */2007-10-27T03:32:56Z<p><span class="autocomment">'''Results'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The probability of lysis given a specific induction of the circuit by tetracycline. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* The probability of lysis given a specific induction of the circuit by tetracycline. </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>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|<del class="diffchange diffchange-inline">300px</del>|<del class="diffchange diffchange-inline">left</del>|Schematic of modeling output]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The modeling exercise provided us a proof of principle that it should be possible to engineer production of lambda repressor under the control of tetracycline such that tetracycline resistant cells are under a fitness disadvantage regime and are out competed by the tetracycline sensitive cells. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Before, the full scale simulation was run, preliminary models were run in order to better understand the results of the different plasmid constructs. These experimental results helped to fine tune the parameters of the model. This refined model could then predict the dynamic of the engineered circuit within the phage genome in order to understand the effects of stochasticity and gene copy number on the efficacy of the circuit's ability to decrease the fitness of resistant cells. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">In the early stages of designed the circuit, the negative feedback on TetR expression was added in order to decease the noise in the circuit and to provide more stable expression levels of TetR for varying gene copy numbers as predicted by the preliminary model. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Later, we used the model to analyze the effects of altering the strength of cI repression by TetR as well as the negative feedback of TetR.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:modeling_output_schematic.jpg|thumb|'''modeling_output_schematic'''|<ins class="diffchange diffchange-inline">400px</ins>|<ins class="diffchange diffchange-inline">right</ins>|Schematic of modeling output]]</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">[[Image:HighPlasmidMeanEyfp.jpg.jpg|thumb|'''HighPlasmidMeanEyfp'''|400px|right|Difference between Single Cell Fluorescence levels in Tet sensitive versus resistant cells ]]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </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>==== '''Characterization of the Circuit''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Characterization of the Circuit''' ====</div></td></tr>
</table>Bibhashhttp://2007.igem.org/wiki/index.php?title=Rice/Project_A:_Phage_Project&diff=47694&oldid=prevTstevenson: /* '''Goals''' */2007-10-27T03:28:27Z<p><span class="autocomment">'''Goals'''</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Goals''' ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== '''Goals''' ====</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>By conferring selective advantage on bacterial population, we intend to artificially create changes in evolutionary fitness landscape <del class="diffchange diffchange-inline">such </del>that bacteria harboring antibiotic resistance are competed <del class="diffchange diffchange-inline">out</del>. <del class="diffchange diffchange-inline">Towards this, we </del>will take advantage of the following: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>By conferring selective advantage on <ins class="diffchange diffchange-inline">a </ins>bacterial population, we intend to artificially create changes in <ins class="diffchange diffchange-inline">the </ins>evolutionary fitness landscape <ins class="diffchange diffchange-inline">so </ins>that bacteria harboring antibiotic resistance are <ins class="diffchange diffchange-inline">out-</ins>competed. <ins class="diffchange diffchange-inline">We </ins>will take advantage of the following: </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Antibiotic as a signaling molecule </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Antibiotic as a signaling molecule </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. 'Immunity' of bacterial strain to lambda phage infection</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. 'Immunity' of bacterial strain to lambda phage infection</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>4. <del class="diffchange diffchange-inline">Correlate </del>sensitivity to antibiotic concentration with lysis versus lysogeny decision in phage infected bacteria.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>4. <ins class="diffchange diffchange-inline">Correlation of </ins>sensitivity to antibiotic concentration with lysis versus lysogeny decision in phage infected bacteria.</div></td></tr>
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<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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== '''Experimental System and Circuit Design''' ==</div></td></tr>
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</table>Tstevenson