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Team:RHIT/Modeling
From 2012.igem.org
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| - | + | <p>The first step of the modeling process was to gain an understanding of the various chemical and biological agents, how they interacted with each other, and ultimately how these interactions caused the physiological changes in the cell. In order to accomplish this, the team spent several hours doing research on the various proteins involved in the pathway. Once the key components were identified, a simplified explanation of the process of interest was created. Below is the mechanism the team used for the basis of the model:</p><br /> | |
| + | <li>Mating Factor Binds to Ste2/Ste3 homodimer (Ste2 for alpha factor / Ste3 for a factor)</li> | ||
| + | <li>Gpa1 subunit exchanges GDP for GTP</li> | ||
| + | <li>Gpa1 releases Ste4/G-gamma heterodimer</li> | ||
| + | <li>Ste4/Ste18 (through Ste4), transmits the signal to the following complexes, Ste5/Ste11, Ste20 protein kinase, and a Far1/Cdc24 complex</li> | ||
| + | <li>Ste4 binds to Cdc24, Far1 brings Cdc24 to Cdc42, Cdc24 facilitates the exchange of GDP for GTP, causing activated Cdc42 to bind to the complex between Ste20 and Bem1, resulting in the activation of Ste20 | ||
| + | * Ste20 is in a low-activity state before being bound by Cdc42</li> | ||
| + | <li>Ste4 binds to Ste5, Ste5 acts as an adaptor to bring Ste4-beta and Ste11 toward Ste20</li> | ||
| + | <li>Ste20 phosphorylates Ste11 (aided by Ste50), causing it to activate</li> | ||
| + | <li>Ste11 then activates Ste7 by phosphorylating it</li> | ||
| + | <li>Ste7 in turn activates Fus3 and Kss1 by phosphorylating them | ||
| + | *Ste5 also plays a role in all both of these events</li> | ||
| + | <li>Fus3 and Kss1 phosphorylate Ste12, Dig1 and Dig2, in their phosphorylated state Dig1 and Dig2 release activated Ste12 | ||
| + | *Kss1 holds the Dig1/Dig2 complex together with Ste12</li> | ||
| + | <li>Activated Ste12 binds to DNA to facilitate transcription of pheromone response genes</li><br /> | ||
| + | <p>After laying out this process, the team began making evaluations as to which parts of the process would be important, and what kind of questions could be answered by the model. After careful deliberation and discussion, the team decided that the questions of interest to the project were to determine the sensitivity of the circuit and the time after exposure that fluorescence is detectable. With those questions in mind, the system was broken into four distinct portions: initiation, transduction, production, and regulation.</p> | ||
| + | <p>The initiation portion of the model pertained to the interactions between the pheromone and the receptor. Since one of the goals of the model was to address the sensitivity of the construct, this portion was mostly conserved and incorporated into the model. The transduction portion covered most of the unmodified kinase cascade covered in the mechanism. Because of the relatively short time period of these interactions in comparison to the approximated time frame of the circuit, and the well characterized and unchanged nature of this part of the pathway, these interactions were removed from the model. The third portion of the model covered the transcriptional and translational activities necessary for the production of the construct. The final module of the model was accounting for the regulatory functions contained in the construct.</p> | ||
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Revision as of 14:45, 1 August 2012
