Team:LCG-UNAM-Mexico/Notebook/2008-July
From 2008.igem.org
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong><br> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong><br> |
We read chapter 2 of Predictive Microbiology by T.A. McMeekin et al., 1993. It taught us the standard stages of a modeling process, the purpose of mathematical models.<br> | We read chapter 2 of Predictive Microbiology by T.A. McMeekin et al., 1993. It taught us the standard stages of a modeling process, the purpose of mathematical models.<br> | ||
The suggested stages of the modeling process are:<br> | The suggested stages of the modeling process are:<br> | ||
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- Validation and maintenance (Including Function Fitting, Model Validation and Model comparisons)</p> | - Validation and maintenance (Including Function Fitting, Model Validation and Model comparisons)</p> | ||
<p>Remarkably this chapter also mentioned the changes of the conductivity of the medium depending on the cellular metabolism, therefore pointing out the importance of having a cellular population which isn’t growing; this to prevent the noise and minimize the conductance change due to bacterial metabolism. </p> | <p>Remarkably this chapter also mentioned the changes of the conductivity of the medium depending on the cellular metabolism, therefore pointing out the importance of having a cellular population which isn’t growing; this to prevent the noise and minimize the conductance change due to bacterial metabolism. </p> | ||
- | <p><strong> | + | <p><strong><u>WET LAB:</u></strong><br></p> |
- | <p><strong | + | <p><strong>Extraction</strong></p> |
<p>Plasmid DNA extraction finished</p> | <p>Plasmid DNA extraction finished</p> | ||
- | <p | + | <p><strong>Bioparts & Transformation</strong></p> |
<p>Bioparts (BBa_C0051 and BBa_I729006) transformation in DH5alfa cells using the lab technique.</p> | <p>Bioparts (BBa_C0051 and BBa_I729006) transformation in DH5alfa cells using the lab technique.</p> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong><br> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong><br> |
System to be analyzed, taken from by Iwig et al. (2006):</p> | System to be analyzed, taken from by Iwig et al. (2006):</p> | ||
<p align="center"<img src="https://static.igem.org/mediawiki/igem.org/d/db/Iwig_2006.jpg" alt="Iwig 2006" /></p> | <p align="center"<img src="https://static.igem.org/mediawiki/igem.org/d/db/Iwig_2006.jpg" alt="Iwig 2006" /></p> | ||
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<p> | <p> | ||
We also defined the variables and equations for our first approach and built our stoichiometric matrix. This can be found in the modeling section. </p> | We also defined the variables and equations for our first approach and built our stoichiometric matrix. This can be found in the modeling section. </p> | ||
- | <p><strong> | + | <p><strong><u>WET LAB</u>:</strong><br></p> |
- | <p><strong | + | <p><strong>Bioparts</strong></p> |
<p>Transformation of bioparts (BBa_C0051 and BBa_I729006) in TOP10 cells using the iGEM protocol.(No one cell grew in the DH5alfa transformation from day 3)</p> | <p>Transformation of bioparts (BBa_C0051 and BBa_I729006) in TOP10 cells using the iGEM protocol.(No one cell grew in the DH5alfa transformation from day 3)</p> | ||
- | <p><strong | + | <p><strong>Plasmids</strong></p> |
<p>Plasmid digestions using the following "recipes":</p> | <p>Plasmid digestions using the following "recipes":</p> | ||
<table border="1" cellspacing="0" cellpadding="0" width="500"> | <table border="1" cellspacing="0" cellpadding="0" width="500"> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING: </strong><br> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u> </strong><br> |
We defined our preliminary model’s assumptions and our equations with their respective stoichiometric matrix. The current assumptions and equations can be found in the Modeling section. </p> | We defined our preliminary model’s assumptions and our equations with their respective stoichiometric matrix. The current assumptions and equations can be found in the Modeling section. </p> | ||
<p>NOTES: <br> | <p>NOTES: <br> | ||
- (d/dt)x=Ʃ(entering flows) - Ʃ(exiting flows)<br> | - (d/dt)x=Ʃ(entering flows) - Ʃ(exiting flows)<br> | ||
- Law of mass action: The flow is equal to the reaction constant times the product of the mass of the reactants. </p> | - Law of mass action: The flow is equal to the reaction constant times the product of the mass of the reactants. </p> | ||
- | <p>TO-DO LIST: <br> | + | <p><u><b>TO-DO LIST: </b></u><br> |
-Start doing simulations with Gepasi, Copasi or Matlab. <br> | -Start doing simulations with Gepasi, Copasi or Matlab. <br> | ||
-Investigate the underlying dynamic of each equation (Law of Mass Action, Hill, Michaelis-Menten, etc.)<br> | -Investigate the underlying dynamic of each equation (Law of Mass Action, Hill, Michaelis-Menten, etc.)<br> | ||
-Read the articles on our webpage, especially Adam Arkin’s and the one regarding lambda phage. </p> | -Read the articles on our webpage, especially Adam Arkin’s and the one regarding lambda phage. </p> | ||
- | <p><strong> | + | <p><strong><u>WET LAB:</u></strong><br></p> |
- | <p><strong | + | <p><strong>Gel</strong></p> |
<p>We run a Gel to observe the digested plasmids. </p> | <p>We run a Gel to observe the digested plasmids. </p> | ||
<p><img src="https://static.igem.org/mediawiki/2008/f/fe/Gel_04Jul08.png" alt="Gel_04Jul08" width="400" /></p> | <p><img src="https://static.igem.org/mediawiki/2008/f/fe/Gel_04Jul08.png" alt="Gel_04Jul08" width="400" /></p> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong></p> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong></p> |
<p>MISSING REACTION:<br> | <p>MISSING REACTION:<br> | ||
We forgot to take into account one reaction, cI* degradation (cI* -» 0). Now it has been added to the model. </p> | We forgot to take into account one reaction, cI* degradation (cI* -» 0). Now it has been added to the model. </p> | ||
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<p>MODEL:<br> | <p>MODEL:<br> | ||
The first simulation in Matlab was generated. It worked! Everyone should test it and correct or suggest anything necessary.</p> | The first simulation in Matlab was generated. It worked! Everyone should test it and correct or suggest anything necessary.</p> | ||
- | <p>TO-DO LIST:<br> | + | <p><u><b>TO-DO LIST:</u></b><br> |
Defining parameters is our topmost priority (reaction rates, kinetic constants and concentrations). </p></div></td> | Defining parameters is our topmost priority (reaction rates, kinetic constants and concentrations). </p></div></td> | ||
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- | <td class="bodyText"><div align="justify"><p>Today we discussed the following with Miguel:</p> | + | <td class="bodyText"><div align="justify"><p><b><u>GROUP MEETING:</b></u><br>Today we discussed the following with Miguel:</p> |
<p><strong>BIOPARTS:</strong><br> | <p><strong>BIOPARTS:</strong><br> | ||
There was information in regards to the transformation of the bioparts. Chiba’s team explained to us that their biopart’s DNA was not in the registry however it was mistakenly set as available. However they assured us that they have the functional bioparts, so we will have to request some DNA directly to the team.</p> | There was information in regards to the transformation of the bioparts. Chiba’s team explained to us that their biopart’s DNA was not in the registry however it was mistakenly set as available. However they assured us that they have the functional bioparts, so we will have to request some DNA directly to the team.</p> | ||
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<p><strong>CONTROLS:</strong><br> | <p><strong>CONTROLS:</strong><br> | ||
Now that the experimental work has inevitably been delayed, that time can be invested in defining controls, both positive and negative for the experiment. </p><br> | Now that the experimental work has inevitably been delayed, that time can be invested in defining controls, both positive and negative for the experiment. </p><br> | ||
- | <p><strong>MODELING:</strong><br> | + | <p><strong><u>MODELING:</u></strong><br> |
Defining parameters:<br> | Defining parameters:<br> | ||
Obtaining the kinetic parameters experimentally is impossible right now; we first need the working bioparts. Right now we could get an average half-life of other proteins, which share characteristics with our desired protein (for example, average half-life of membrane proteins to define RcnA’s half-life) and trust that they won’t differ much. Once the experimental work resumes we could compare our parameters with those obtained experimentally. </p> | Obtaining the kinetic parameters experimentally is impossible right now; we first need the working bioparts. Right now we could get an average half-life of other proteins, which share characteristics with our desired protein (for example, average half-life of membrane proteins to define RcnA’s half-life) and trust that they won’t differ much. Once the experimental work resumes we could compare our parameters with those obtained experimentally. </p> | ||
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<li>Null is a special variable of the program to define the degradation of a protein, the model was corrected to use this variable. </li> | <li>Null is a special variable of the program to define the degradation of a protein, the model was corrected to use this variable. </li> | ||
</ol> | </ol> | ||
- | <p>TO-DO LIST:<br> | + | <p><u><b>TO-DO LIST:</u></b><br> |
Parameters: We are going to focus on this. </p> | Parameters: We are going to focus on this. </p> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong></p> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong></p> |
<p>Parameters to be defined:<br> | <p>Parameters to be defined:<br> | ||
Half-lives, Promoter efficiency, Dissociation rates and Initial concentrations</p> | Half-lives, Promoter efficiency, Dissociation rates and Initial concentrations</p> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong></p> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong></p> |
<p><strong>Michaelis-Menten Kinetics</strong><br> | <p><strong>Michaelis-Menten Kinetics</strong><br> | ||
We read Lehninger Principles of Biochemistry (Many Enzymes Catalyze Reactions with Two or More Substrates, pp.207) to better understand Michaelis-Menten Kinetics. Mariana summarized and condensed the information in a document for everyone else to read. </p></div></td> | We read Lehninger Principles of Biochemistry (Many Enzymes Catalyze Reactions with Two or More Substrates, pp.207) to better understand Michaelis-Menten Kinetics. Mariana summarized and condensed the information in a document for everyone else to read. </p></div></td> | ||
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- | <td class="bodyText"><div align="justify"><p><strong>MODELING:</strong><br> | + | <td class="bodyText"><div align="justify"><p><strong><u>MODELING:</u></strong><br> |
We checked the dynamics of our equations further developing the beforementioned document<br> | We checked the dynamics of our equations further developing the beforementioned document<br> | ||
Reminder:<br> | Reminder:<br> | ||
According to Michaelis-Menten: V0=[kcat*Et*S]/[km+S] where Vmax=kcat*Et (as long as the enzyme is saturated).</p> | According to Michaelis-Menten: V0=[kcat*Et*S]/[km+S] where Vmax=kcat*Et (as long as the enzyme is saturated).</p> | ||
- | <p>TO-DO LIST:<br> | + | <p><u><b>TO-DO LIST:</u></b><br> |
- Find missing parameters and start evaluating the model<br> | - Find missing parameters and start evaluating the model<br> | ||
- Add the dimerization of cI* and of the complex AHL:LuxR to the model<br> | - Add the dimerization of cI* and of the complex AHL:LuxR to the model<br> | ||
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- | <td class="bodyText"><div align="justify"><p> <strong>MODELING:</strong><br> | + | <td class="bodyText"><div align="justify"><p> <strong><u>MODELING:</u></strong><br> |
Analyzed Hill Coefficient.</p> | Analyzed Hill Coefficient.</p> | ||
- | <p>TO-DO LIST:<br> | + | <p><u><b>TO-DO LIST:</u></b><br> |
- Understand Hill Cooperativity<br> | - Understand Hill Cooperativity<br> | ||
- Find missing parameters<br> | - Find missing parameters<br> | ||
- Determine initial Concentrations<br> | - Determine initial Concentrations<br> | ||
- Include Dimerization</p> | - Include Dimerization</p> | ||
- | <p><strong> | + | <p><strong><u>WET LAB:</u></strong><br></p> |
- | <p><strong | + | <p><strong><span bold="">Cultures</span></strong></p> |
<p>We cultured the cells with the plasmids</p></div></td> | <p>We cultured the cells with the plasmids</p></div></td> | ||
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- | <td class="bodyText"><div align="justify"><p><strong> | + | <td class="bodyText"><div align="justify"><p><strong><u>WET LAB:</u></strong><br></p> |
- | <p><strong | + | <p><strong>Transformación</strong></p> |
<p>We transformed the biopart from Chiba(BBa_I729006) using two controls:</p> | <p>We transformed the biopart from Chiba(BBa_I729006) using two controls:</p> | ||
<p>*Control 1: only DH5 alfa cells<br /> | <p>*Control 1: only DH5 alfa cells<br /> | ||
*Control 2: without cells</p> | *Control 2: without cells</p> | ||
- | <p><strong | + | <p><strong>PCR</strong></p> |
<ol> | <ol> | ||
<li>Part 1 from Chiba's Biopart</li> | <li>Part 1 from Chiba's Biopart</li> | ||
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- | <td class="bodyText"><div align="justify"><p>The cell colonies transformed with the biopart from Chiba(BBA_I729006), grew successfully.<br /> | + | <td class="bodyText"><div align="justify"><p><b><u>WET LAB:</u></b><br>The cell colonies transformed with the biopart from Chiba(BBA_I729006), grew successfully.<br /> |
Three colonies from each cage were plated(concentred and no concentred), in 5 mL of liquid LB.</p> | Three colonies from each cage were plated(concentred and no concentred), in 5 mL of liquid LB.</p> | ||
<p>We run a with the PCR products.</p> | <p>We run a with the PCR products.</p> | ||
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- | <td class="bodyText"><div align="justify">< | + | <td class="bodyText"><div align="justify"><u><b>WET LAB:</u></b><br><p><strong>PCR</strong></p> |
<p>We performed the PCR of the missing tubes</p> | <p>We performed the PCR of the missing tubes</p> | ||
- | <p><strong | + | <p><strong>Purification of PCR Product</strong></p> |
<p><img src="https://static.igem.org/mediawiki/2008/6/64/Gel_25Jul08.png" alt="Gel_25Jul08" width="400"/></p> | <p><img src="https://static.igem.org/mediawiki/2008/6/64/Gel_25Jul08.png" alt="Gel_25Jul08" width="400"/></p> | ||
</div></td> | </div></td> | ||
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- | <td class="bodyText"><div align="justify"><p><strong><u> | + | <td class="bodyText"><div align="justify"><p><strong><u>WET LAB:</u></strong></p><b>Gel</b><br> |
<p>We run a gel in order to observe the PCR products of the 25th.</p> | <p>We run a gel in order to observe the PCR products of the 25th.</p> | ||
<p><img src="https://static.igem.org/mediawiki/2008/2/2c/Gel_28Jul08_copy.png" alt="Gel_28Jul08" width="400" /></p> | <p><img src="https://static.igem.org/mediawiki/2008/2/2c/Gel_28Jul08_copy.png" alt="Gel_28Jul08" width="400" /></p> | ||
- | <p><strong | + | <p><strong>Restrictions(cuts)</strong></p> |
<p>EcoR1-Up/Bam-Lowe Parte1 (10 μl of PCR sample)<br /> | <p>EcoR1-Up/Bam-Lowe Parte1 (10 μl of PCR sample)<br /> | ||
Xba1-Up/Pst1-Low Parte3-N (8 μl of PCR sample)<br /> | Xba1-Up/Pst1-Low Parte3-N (8 μl of PCR sample)<br /> | ||
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- | <td class="bodyText"><div align="justify"><p><strong><u> | + | <td class="bodyText"><div align="justify"><p><strong><u>WET LAB:</u></strong></p><b>Cultures</b><br> |
<p>From each one of the samples cultured on 28th,we chose 5 colonies and we plated in a 1 mL LB tube and we incubate it for 6 hrs. Our neative control was just LB.</p> | <p>From each one of the samples cultured on 28th,we chose 5 colonies and we plated in a 1 mL LB tube and we incubate it for 6 hrs. Our neative control was just LB.</p> | ||
<p><strong><u>Bioparts ligation in pRK415 and pBB</u></strong></p> | <p><strong><u>Bioparts ligation in pRK415 and pBB</u></strong></p> | ||
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</table> | </table> | ||
- | <p><strong | + | <p><strong>Transformation</strong></p> |
<p>We transformed the product of the ligation and plated it in cages with X-gal. With he purpose of save X-gal we chose to add it not in the LB-agar, but joint to the plated culture. In order to do this, we dilute 20 mL of X-Gal in 1mL of N-N-dimethylformamide. And from this mix we plated 25μl in each cage.</p> | <p>We transformed the product of the ligation and plated it in cages with X-gal. With he purpose of save X-gal we chose to add it not in the LB-agar, but joint to the plated culture. In order to do this, we dilute 20 mL of X-Gal in 1mL of N-N-dimethylformamide. And from this mix we plated 25μl in each cage.</p> | ||
<p>For the transformation we used two controls( one without plasmid and one without cell). When plating we excluded the control without cell and plated the DH5 cells without transformation in a Tc Cage and another of Gm.</p> | <p>For the transformation we used two controls( one without plasmid and one without cell). When plating we excluded the control without cell and plated the DH5 cells without transformation in a Tc Cage and another of Gm.</p> | ||
- | <p><strong | + | <p><strong>Plasmid Extraction</strong></p> |
<p>We extract plasmid from te plated colonies. All the tubes pass the night with 1mL of ethanol at 100%.</p> | <p>We extract plasmid from te plated colonies. All the tubes pass the night with 1mL of ethanol at 100%.</p> | ||
- | <p><strong | + | <p><strong>Biopart cI</strong></p> |
<p>The missing biopart(cI), was cultured in 5mL of LB with 2.5 μl of ampicillin(50%). It was also plated in 5mL of LB without antibiotic and was plated in agar with ampicillin at 100%</p> | <p>The missing biopart(cI), was cultured in 5mL of LB with 2.5 μl of ampicillin(50%). It was also plated in 5mL of LB without antibiotic and was plated in agar with ampicillin at 100%</p> | ||
</div></td> | </div></td> | ||
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- | <td class="bodyText"><div align="justify"><p><strong><u> | + | <td class="bodyText"><div align="justify"><p><strong><u>WET LAB:</u></strong></p><b>Plasmid extraction</b><br> |
<p>We finished the extraction of the plasmid, started on 29th.<br /> | <p>We finished the extraction of the plasmid, started on 29th.<br /> | ||
We extracted the plasmid holding the biopart cI.</p> | We extracted the plasmid holding the biopart cI.</p> | ||
- | <p><strong | + | <p><strong>Gel</strong></p> |
<p>We run a Gel with the 15 samples of the plasmid pHET containing RcnA, part 3(normal) or part 3 (mut) and the three extraction plasmid samples containing the biopart cI+LVA</p></div></td> | <p>We run a Gel with the 15 samples of the plasmid pHET containing RcnA, part 3(normal) or part 3 (mut) and the three extraction plasmid samples containing the biopart cI+LVA</p></div></td> | ||
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