http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Patsmad&year=&month=2009.igem.org - User contributions [en]2024-03-29T12:59:02ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Minnesota/ModelingTeam:Minnesota/Modeling2009-08-05T15:53:44Z<p>Patsmad: </p>
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<h2>Our Modeling Goals</h2><br />
[[Image:ModelingLab2.jpg|300px|right|The modelers in their natural environment.]]<br />
<br />
The primary goal of the modeling group is to create simulations of our experimental results. Our computational output will guide future research and helps us to understand what is happening at the molecular level of our logical AND gate.<br />
It is important to be able to observe the fidelity of the AND gate <i>in silico</i> as well as <i>in vivo</i>. Using our in-house software suite, SynBioSS, we will create reaction networks describing the AND gate behavior with different constructs (e.g. TTN, TTL, TNN) and mutations. These reaction networks can then used to create the files necessary for simulation on the supercomputer. Certain kinetic constants in the reaction network can be adjusted to fit the simulation to experimental data. By observing which kinetic constants change with AND-gate construction or mutation we will then be able to understand some of the ways in which the AND-gate functions.<br />
<br />
<h2>Mathematical Modeling</h2><br />
<h3>Overview</h3><br />
[[Image:designgui.jpeg|300px|left|thumb|The MATLAB GUI displays all the reactions and chemical species in the network and allows the user to manipulate all aspects of the system such as reaction rates in order to better mimic experimental results.]]<p>In order to create a model, we must first use a program that will allow us to enter in the contents of the reactions network. The reaction network is formatted as .nc file, which is a special format called NetCDF that can interact with the group's supercomputer algorithms. The reaction network can be inputted directly into Linux as a text file and then converted into an .nc file. However, this is very tedious and error-prone process. In order to more quickly and accurately create the models, we use one of two programs: SynBioSS or a MATLAB GUI. Each of these programs allows us to graphically see what the model looks like and automatically converts the data describing the system into an .nc file. (See screenshots). <br> <br />
Once the model is created, it is sent to a supercomputer where it is run using hybrid stochastic simulation algorithms created by the Kaznessis group. This algorithm takes the initial conditions of the model along with the reactions and kinetic constants and determines the state of the system over time. A stochastic model is used because the system that is described is very small. Cells are typically on the order of 1E-15 L. Because of this many molecular species exist in small quantities. When there are low numbers of molecules in a system, discrete changes in the number of molecules drastically change the reaction rates. Stochastic models use random number generators to describe the probabilistic nature of stochastic systems. <br><br />
The results of the models are then retrieved from the supercomputer and analyzed using MATLAB. It is then possible to see if the model ran correctly and if it gave reasonable results. Depending on how the model compares to the experimental data, kinetic constants are changed and then the process of running a model begins again.<br><br />
The accuracy of the model is determined by the difference in reporter gene expression between the model and the experiments. In order to make reasonable comparison the models must be rescaled, so that the model data matches with the experimental numerically. Basal expression models, which produce the maximum amount of reporter gene, are created for both the experimental and simulation data. This is done by removing the repressor molecules tetR2 and lacR4 from the system. Using this data, it is possible to rescale the model and the experimental data as a percentage of maximum expression. A least squares regression can then be done to determine the difference between the experimental results and the simulation. <br><br />
Once the simulation is as close to the experimental results as possible a more accurate description of the AND-gate function can be constructed. This can be done by observing how changes in kinetic constants affect GFP expression. Or, using experimental data for mutant constructs, the simulations can be re-fit and the mutation's affect on the reaction network can be quantified. </p><br />
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<h2>Some Basic Modeling</h2><br />
[[Image:reactionlist.jpg|200px|right|The reaction list in the MATLAB GUI with reactions 3 and 54 highlighted.]]<br />
<br />
We are working with four different combinations of <i>lac</i> and<i>tet</i> (L and T, respectively) operators in our promoter design project. These constructs are: T--, TT-, TTL and -T-. Here, we will demonstrate a little basic modeling we have done of the TTL construct using SynBioSS MATLAB and a design GUI. <br />
<br />
On the SynBioSS wiki, the TTL model is available for download in a .nc format. Upon opening this with the MATLAB GUI, the list of 65 reactions that constitute the repression and induction of transcription. In this model, there are two <i>tet</i> operators TO1 and TO2 and one <i>lac</i> operator LO1. The reactions included in this list account for the repression and induction of transcription in the absence and presence of aTc and IPTG, respectively. In this simple model, we examined reactions 3 and 54, which involve the <i>tet</i> operators being bound by the repressor. (These reactions are shown in the GUI on the left.) We varied the reactions constants from the original 10<sup>8</sup> as low as 0 and as high as 10<sup>9</sup> to see if the fidelity of the AND gate could depend on the reaction rates for repression. We quantified our results by counting molecules of GFP. This not only gave us a clearer idea of what was happening at the molecular level at this promoter site, but also served as a preparation for matching the experimental results computationally.<br />
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<h2>Calendar</h2><br />
{| align="center"<br />
{| style="color:gold;background-color:#800000;" cellpadding="3" cellspacing="1" border="10" bordercolor="#00CCFF" width="62%" align="center"<br />
!align="center"|{{#calendar: title=Minnesota |year=2009 | month=06}}<br />
!align="center"|{{#calendar: title=Minnesota |year=2009 | month=07}}<br />
!align="center"|{{#calendar: title=Minnesota |year=2009 | month=08}}<br />
!align="center"|{{#calendar: title=Minnesota |year=2009 | month=09}}<br />
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<h2>Results</h2><br />
Place results of models here.</div>Patsmadhttp://2009.igem.org/Minnesota/9_June_2009Minnesota/9 June 20092009-08-02T16:26:37Z<p>Patsmad: </p>
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Today Patrick arrived and began working on getting up to speed on the team's TTL model and creating the TTN model he will eventually be working on.<br><br />
The model created was similar to that described previously except with the addition of a second tet operon (tetO2):<br><br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Rxn #<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|1/2||RNAp + lacP + tetO1 + tetO2 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|3||RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|4||RNAp:lacP* -> lacP + tetO1 + tetO2 + RNAp:DNAgfp||30||<br />
|-<br />
|5||RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|6||gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|7||rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|8||rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|9||gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|10||gfp -> Ø||3.21E-05||<br />
|-<br />
|11\12||tetR2 + aTc ↔ tetR2:aTc||1E+08||1E-03<br />
|-<br />
|13\14||tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|15\16||tetR2 + tetO1 ↔ tetR2:tetO1||1E+08||1E-03<br />
|-<br />
|17\18||tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|19\20||tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|21\22||tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|23\24||tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|25\26||tetR2 + tetO2 ↔ tetR2:tetO2||1E+08||1E-03<br />
|-<br />
|27\28||tetR2:aTc + tetO2 ↔ tetR2:tetO2:aTc||1E+08||1<br />
|-<br />
|29\30||tetR2:aTc2 + tetO2 ↔ tetR2:tetO2:aTc2||1E+08||1E+05<br />
|-<br />
|31\32||tetR2:tetO2 + aTc ↔ tetR2:tetO2:aTc||1E+08||1E-03<br />
|-<br />
|33\34||tetR2:tetO2:aTc + aTc ↔ tetR2:tetO2:aTc2||1E+08||1E-03<br />
|-<br />
|35||tetR2 -> Ø||2.89E-04||<br />
|-<br />
|36||tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|37||tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|38\39||tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|40\41||tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|42||tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|43||tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|44||Ø -> tetR2||1E-11||<br />
|-<br />
|45||Ø -> aTc||3.3E-04||<br />
|}<br />
<br>Since we needed to have the aTc concentration constant both of the models created eliminated the aTc in all the equations and physically set each kinetic constant according to the desired aTc concentration.</div>Patsmadhttp://2009.igem.org/Minnesota/30_June_2009Minnesota/30 June 20092009-07-30T22:19:19Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
Here is the "leaky" graph along with the base for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:badBase.jpg|Figure 1 - Base TTN Model<br />
Image:Leak.jpg|Figure 2 - Leaky Model<br />
</gallery></center><br><br />
<br />
As can be seen the "leaky" graph indeed does show increased GFP production at low aTc concentrations.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky11.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/29_June_2009Minnesota/29 June 20092009-07-30T22:17:24Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
I switched over today to looking at making the "leakiness" equations for the TTN model. Initially I would represent the leakiness with two equations:<br><br />
<br />
<center><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Rxn #<br />
! Reaction<br />
! Forward Kinetic Constant<br />
|-<br />
|46||RNAp + lacP + tetO1:tetR2 + tetO2 -> RNAp:lacP||1E+07<br />
|-<br />
|47||RNAp + lacP + tetO1 + tetO2:tetR2 -> RNAp:lacP||1E+07<br />
|}</center><br><br />
<br />
The aTc complexes don't have to be represented since the leakiness is for aTc=0 in particular. Also a third leakiness equation could be made, one where both tetO1:tetR2 and tetO2:tetR2 exist, but I chose to add this later if necessary. I expect the results tomorrow.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky9.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/29_June_2009Minnesota/29 June 20092009-07-30T22:17:03Z<p>Patsmad: </p>
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<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
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|'''[[Minnesota/26 June 2009|Go to Previous Day (June 26)]]'''|| width=158|'''[[Minnesota/30 June 2009|Go to Next Day (June 30)]]'''<br />
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'''Patrick'''<br><br />
I switched over today to looking at making the "leakiness" equations for the TTN model. Initially I would represent the leakiness with two equations:<br><br />
<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Rxn #<br />
! Reaction<br />
! Forward Kinetic Constant<br />
|-<br />
|46||RNAp + lacP + tetO1:tetR2 + tetO2 -> RNAp:lacP||1E+07<br />
|-<br />
|47||RNAp + lacP + tetO1 + tetO2:tetR2 -> RNAp:lacP||1E+07<br />
|}<br><br />
<br />
The aTc complexes don't have to be represented since the leakiness is for aTc=0 in particular. Also a third leakiness equation could be made, one where both tetO1:tetR2 and tetO2:tetR2 exist, but I chose to add this later if necessary. I expect the results tomorrow.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky9.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/29_June_2009Minnesota/29 June 20092009-07-30T22:13:25Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
I switched over today to looking at making the "leakiness" equations for the TTN model. Initially I would represent the leakiness with two equations:<br><br />
<br />
<br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky9.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/26_June_2009Minnesota/26 June 20092009-07-30T22:08:53Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
I got back the analysis of '''tetRu/d''' which looked at the effects of increasing or decreasing the rate of synthesis of tetR2 (reaction 44, k = 1E-11 M/s initially) by an order of magnitude). Here are the results along with the base model for comparison:<br><br />
<br />
<center><gallery widths=250 heights=200><br />
Image:badBase.jpg|Figure 1 - Base TTN Model<br />
Image:badTetRu.jpg|Figure 2 - Increased TetR2 Synthesis<br />
Image:badTetRd.jpg|Figure 3 - Decreased TetR2 Synthesis<br />
</gallery></center><br><br />
<br />
These results look more promising as they shift the "hump" dramatically. The "hump" is an issue though since GFP production should reach steady state in 9 hours.<br><br />
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'''Ben'''<br><br />
This model (tnn_atcvaries7_leaky) was an attempt to see what would happen if the kinetic constant was decreased for the three reactions shown below, instead of increased like the previous day's. <br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||31000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky7.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
The result of this change was quite interesting. The curve at aTc = 0 remained constant, while the entire rest of the model has been lowered. This precisely the opposite of what is needed for the model unfortunately. This direction will probably not be pursued in the future as it is not helping solve the porblems seen in the model.</div>Patsmadhttp://2009.igem.org/Minnesota/25_June_2009Minnesota/25 June 20092009-07-30T22:04:28Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
I got back the analysis of the models submitted yesterday. I submitted a base model shown here:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:badBase.jpg|Figure 1 - Base TTN Model<br />
</gallery></center><br><br />
<br />
I also changed the tendency for tetO1 to detach from the tetR2:tetO1:2aTc complex (k=100000 1/s initially) by lower or higher an order of magnitude:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:badk2u.jpg|Figure 2 - Increased Rate of tetO1 Detachment from tetR2:tetO1:2aTc complex<br />
Image:badk2d.jpg|Figure 3 - Decreased Rate of tetO1 Detachment from tetR2:tetO1:2aTc complex<br />
</gallery></center><br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:badk2u.jpg|Figure 4 - Increased Rate of tetO1 Detachment from tetR2:tetO1:aTc complex<br />
Image:badk2d.jpg|Figure 5 - Decreased Rate of tetO1 Detachment from tetR2:tetO1:aTc complex<br />
</gallery></center><br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:badk12u.jpg|Figure 6 - Increased Rate of tetO1 Detachment from tetR2:tetO1:2aTc/aTc complex<br />
Image:badk12d.jpg|Figure 7 - Decreased Rate of tetO1 Detachment from tetR2:tetO1:2aTc/aTc complex<br />
</gallery></center><br><br />
<br />
None of the graphs were particularly interesting. I also submitted two more jobs: '''smad_tetRu/d''' which either increased or decreased the rate of synthesis of tetR2 (reaction 44, k = 1E-11 M/s initially) by an order of magnitude.<br><br />
<br />
'''Ben'''<br><br />
In order to try to improve the model, the following reaction constants were changed to try to see less leakiness in the model.<br />
<br><br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||1200000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky5.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
This model does indeed show some different leakiness, which is definitely a good thing, although the leakiness is still too high for an accurate model. Still, the problem remains where the model peaks and decreases instead of reaching a steady state.</div>Patsmadhttp://2009.igem.org/Minnesota/24_June_2009Minnesota/24 June 20092009-07-30T21:52:02Z<p>Patsmad: </p>
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'''Patrick'''<br><br />
After discovering the error in the program I wished to submit many of the same programs to try and get back up to speed. First was '''smad_base''' which was the simple base model. '''smad_k2u/d''' changed the kinetic constant for the release of tetO1 by the tetR2:tetO1:2aTc complex (reaction 20, normally k = 100000 1/s). '''smad_k1u/d''' changed the kinetic constant for the release of tetO1 by the tetR2:tetO1:aTc complex (reaction 18, normally k = 1 1/s). '''smad_k12u/d''' did both at the same time. The results are expected tomorrow.<br><br />
<br />
'''Ben'''<br><br />
Since there are several problems with the first TNN model, we are deciding to focus on them one at a time. The first thing to work on is the leakiness problem. In order to deal with this issue, we are including a reaction that allows for the binding of the RNA polymerase to the operator site even if the site is occupied by a tetR. This will allow for some expression at an aTc concentration of 0, because at this point tetR is almost always bound to the tetO operator.<br />
<br />
Here is the model we are working on:<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|aTc_ext -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
As you can see from the above graph, this model shows no response to aTc concentration. The gfp output is the same whether there are 0 or 260 molecules of aTc in the cell. Essentially this system is completely leaky, which is not what is needed. Also, the model still peaks and decreases, instead of plateauing.</div>Patsmadhttp://2009.igem.org/File:Leak.jpgFile:Leak.jpg2009-07-30T21:46:20Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:BadTetRu.jpgFile:BadTetRu.jpg2009-07-30T21:44:28Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:BadTetRd.jpgFile:BadTetRd.jpg2009-07-30T21:44:23Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk12u.jpgFile:Badk12u.jpg2009-07-30T21:44:16Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk12d.jpgFile:Badk12d.jpg2009-07-30T21:44:10Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk2u.jpgFile:Badk2u.jpg2009-07-30T21:44:02Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk2d.jpgFile:Badk2d.jpg2009-07-30T21:43:56Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk1u.jpgFile:Badk1u.jpg2009-07-30T21:43:50Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:Badk1d.jpgFile:Badk1d.jpg2009-07-30T21:43:44Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:BadBase.jpgFile:BadBase.jpg2009-07-30T21:43:37Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/Minnesota/24_June_2009Minnesota/24 June 20092009-07-30T21:40:55Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 June 2009|Go to Previous Day (June 23)]]'''|| width=158|'''[[Minnesota/25 June 2009|Go to Next Day (June 25)]]'''<br />
|}<br />
'''Patrick'''<br><br />
<br />
'''Ben'''<br><br />
Since there are several problems with the first TNN model, we are deciding to focus on them one at a time. The first thing to work on is the leakiness problem. In order to deal with this issue, we are including a reaction that allows for the binding of the RNA polymerase to the operator site even if the site is occupied by a tetR. This will allow for some expression at an aTc concentration of 0, because at this point tetR is almost always bound to the tetO operator.<br />
<br />
Here is the model we are working on:<br />
{| class="wikitable" style="text-align:center" border = "1"<br />
|-<br />
! Reaction<br />
! Forward Kinetic Constant<br />
! Reverse Kinetic Constant<br />
|-<br />
| RNAp + lacP + lacI4:lacO1 -> RNAp:lacP||6.23E+05|| <br />
|-<br />
| RNAp + lacP + lacO1 ↔ RNAp:lacP||1E+07|| 1<br />
|-<br />
|RNA:lacP -> RNAp:lacP* || .01 ||<br />
|-<br />
|RNAp:lacP* -> lacP + lacO1 + RNAp:DNAgfp||30||<br />
|-<br />
|RNAp:DNAgfp -> RNAp + gfp_mRNA||30||<br />
|-<br />
|gfp_mRNA + rib -> rib:gfp_mRNA||100000||<br />
|-<br />
|rib:gfp_mRNA -> rib:gfp_mRNA_1 + gfp_mRNA||33||<br />
|-<br />
|rib:gfp_mRNA_1 -> rib + gfp||33||<br />
|-<br />
|tetR2 + aTc ↔ tetR2:aTc||2E+09||4E-04<br />
|-<br />
|tetR2:aTc + aTc ↔ tetR2:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2:aTc + tetO1 ↔ tetR2:tetO1:aTc||1E+08||1<br />
|-<br />
|tetR2:aTc2 + tetO1 ↔ tetR2:tetO1:aTc2||1E+08||1E+05<br />
|-<br />
|tetR2:tetO1 + aTc ↔ tetR2:tetO1:aTc||1E+08||1E-03<br />
|-<br />
|tetR2:tetO1:aTc + aTc ↔ tetR2:tetO1:aTc2||1E+08||1E-03<br />
|-<br />
|tetR2 -> Ø||2.89E-04||<br />
|-<br />
|tetR2:aTc -> aTc||2.89E-04||<br />
|-<br />
|tetR2:aTc2 -> 2 aTc||2.89E-04||<br />
|-<br />
|tetR2 + nsDNA ↔ tetR2:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc + nsDNA ↔ tetR2:aTc:nsDNA||1000||3.2409<br />
|-<br />
|tetR2:aTc:nsDNA -> aTc + nsDNA||1.93E-04||<br />
|-<br />
|tetR2:nsDNA -> nsDNA||1.93E-04||<br />
|-<br />
|Ø -> tetR2||1E-11||<br />
|-<br />
|aTc_ext -> aTc||3.3E-04||<br />
|-<br />
|gfp_mRNA -> Ø||1.16E-03||<br />
|-<br />
|gfp -> Ø||3.21E-05||<br />
|-<br />
|RNAp + tetO1:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
|RNAp + tetO1:aTc:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|-<br />
||RNAp + tetO1:aTc2:tetR2 + lacP -> RNAp:lacP + tetR2||311000||<br />
|}<br />
<br><br />
[[Image:Tnnatcvariesleaky3.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
As you can see from the above graph, this model shows no response to aTc concentration. The gfp output is the same whether there are 0 or 260 molecules of aTc in the cell. Essentially this system is completely leaky, which is not what is needed. Also, the model still peaks and decreases, instead of plateauing.</div>Patsmadhttp://2009.igem.org/Minnesota/30_July_2009Minnesota/30 July 20092009-07-30T21:39:43Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/29 July 2009|Go to Previous Day (July 29)]]'''|| width=158|'''[[Minnesota/31 July 2009|Go to Next Day (July 31)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I mostly tried to organize the calendar more. I got all the graphs I made, put them into Word so that I can easily transfer them around. I also organized the entire reaction network, and made sure I could explain why each thing was the way it was in particular.<br><br />
<br />
I submitted a new model, called '''smad_ttn_setTet'''. This attempts to circumvent the issue with tetR2 not splitting appropriately, by simply setting tetR2 to a value and not eliminating or making it, and shutting off the split on division. This mimics a steady-state condition for tetR2. By moving the tetR2 value I'll be able to definitively test what affect a set amount of tetR2 has on the model.<br></div>Patsmadhttp://2009.igem.org/Minnesota/29_July_2009Minnesota/29 July 20092009-07-30T21:36:01Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/28 July 2009|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/30 July 2009|Go to Next Day (July 30)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I put off the preparations for the paper and presentation in order to produce the mutant graphs from the experimental data. Below are a couple examples. The TTN mutants look like I would expect them to (much different than the TTN data given before), which only increases my suspicion of the main data:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:TTNmut4_IPTG0.jpg|Figure 2 - Mutant TTN (4) Experimental Data<br />
Image:TTNmut5_IPTG0.jpg|Figure 1 - Mutant TTN (5) Experimental Data<br />
</gallery></center><br></div>Patsmadhttp://2009.igem.org/File:TTNmut5_IPTG0.jpgFile:TTNmut5 IPTG0.jpg2009-07-30T21:34:17Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/File:TTNmut4_IPTG0.jpgFile:TTNmut4 IPTG0.jpg2009-07-30T21:33:42Z<p>Patsmad: </p>
<hr />
<div></div>Patsmadhttp://2009.igem.org/Minnesota/29_July_2009Minnesota/29 July 20092009-07-30T21:33:18Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/28 July 2009|Go to Previous Day (July 28)]]'''|| width=158|'''[[Minnesota/30 July 2009|Go to Next Day (July 30)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I put off the preparations for the paper and presentation in order to produce the mutant graphs from the experimental data. Below are a couple examples. The TTN mutants look like I would expect them to (much different than the TTN data given before), which only increases my suspicion of the main data:<br></div>Patsmadhttp://2009.igem.org/Minnesota/28_July_2009Minnesota/28 July 20092009-07-30T21:31:59Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/27 July 2009|Go to Previous Day (July 27)]]'''|| width=158|'''[[Minnesota/29 July 2009|Go to Next Day (July 29)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Since the program ends in two weeks I began getting the results so far organized so a report can be made. Making sure the calendar is updated appropriately, getting all the graphs produced and saved, starting to write introductory sections, and explanations of the reaction network, etc.<br><br />
<br />
Meanwhile I will continue to try and refine the model more, although I think there may be a flaw in the experimental data for TTN since it doesn't level off, which is suspicious.<br></div>Patsmadhttp://2009.igem.org/Minnesota/24_July_2009Minnesota/24 July 20092009-07-30T20:33:49Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 July 2009|Go to Previous Day (July 23)]]'''|| width=158|'''[[Minnesota/27 July 2009|Go to Next Day (July 27)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Got back test4, and also created three new programs. Yesterday I submitted two models, the reduction of tetR2's affinity to aTc (reactions 11 and 13) by one (test4) and two (test5) orders of magnitude to see how that affected the model. The base model is also given for comparison:<br />
<br />
<center><gallery widths=250 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:test4.jpg|Figure 2 - Reduced Kinetic Constant for Reaction 11 and 13, One Order of Magnitude<br />
Image:test5.jpg|Figure 3 - Reduced Kinetic constant for Reaction 11 and 13, two Orders of Magnitude<br />
</gallery></center><br><br />
<br />
As can be seen the results were disappointing. ATc still out-competes everything else at high aTc concentrations it seems.<br><br />
<br />
The new models created were called '''smad_ttn_test6''' which took the above idea to the extreme. In order to see how aTc really affected the model, I decided to make sure cutting it out entirely came out with a logical graph. So reaction 11 and 13 kinetic constants were set to zero.<br><br />
<br />
The two other models were '''smad_ttn_test7''' and '''smad_ttn_test8'''. These were made to solve the problem with the non-diluting tetR2:aTc and tetR2:aTc2 complexes. In this case I eliminated tetR2:aTc and tetR2:aTc2 by changing any reaction containing those species to instead result in tetR2 and aTc. This would allow tetR2 to more accurately divide (although it would sacrifice some accuracy as well, since these complexes do in fact exist). Test8 is similar, but it also checked to see how increasing the decomposition of tetR2 (reaction 35, k=2.89E-04 1/s, increase to k=2.89E-03) would affect this modified model.<br><br />
<br />
<br />
'''Ben'''<br><br />
[[Image:Tnnnew28.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew30.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/27_July_2009Minnesota/27 July 20092009-07-30T20:33:18Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/24 July 2009|Go to Previous Day (July 24)]]'''|| width=158|'''[[Minnesota/28 July 2009|Go to Next Day (July 28)]]'''<br />
|}<br />
'''Patrick'''<br><br />
The results for the last three models submitted came back. The first is test6, which looked at how eliminating the ability for tetR2 to attach to aTc would affect the model. While horribly inaccurate, the hope was to simply confirm the model was working as expected. The base model is provided for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:test6.jpg|Figure 2 - Eliminating the Affect of aTc<br />
</gallery></center><br><br />
<br />
This was somewhat surprising, but not unexplainable. Basically what is happening is that tetR2 is getting around and producing tetR2:aTc and tetR2:aTc2 by first producing tetR2:tetO1 complex. The then favorable reaction at high aTc concentrations it to almost immediately move to tetR2:tetO1:aTc2. This then decomposes to tetR2:aTc2 + tetO1. Thus tetR2 gets stuck as the tetR2:aTc2 complex as it did before. This method is not going to work in any practical manner to solve the problem.<br><br />
<br />
The next two models looked at truly eliminating the tetR2:aTc and tetR2:aTc2 complexes. Test7 does this simply by making tetR2:aTc2 go to tetR2 + 2 aTc, immediately upon formation. Test 8 does the same thing, but also increases the tetR2 decomposition:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:test7.jpg|Figure 3 - Eliminating tetR2:aTc and tetR2:aTc2 complexes<br />
Image:test8.jpg|Figure 4 - Eliminating tetR2:aTc and tetR2:aTc2 complexes and Increasing tetR2 Decomposition<br />
</gallery></center><br><br />
<br />
This has a more promising effect. It makes the difference between different aTc concentrations much more apparent. The experimental results somewhat support this idea. Both of these seem like it could work.<br></div>Patsmadhttp://2009.igem.org/Minnesota/24_July_2009Minnesota/24 July 20092009-07-30T20:21:45Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/23 July 2009|Go to Previous Day (July 23)]]'''|| width=158|'''[[Minnesota/27 July 2009|Go to Next Day (July 27)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Got back test4, and also created three new programs. Yesterday I submitted two models, the reduction of tetR2's affinity to aTc (reactions 11 and 13) by one (test4) and two (test5) orders of magnitude to see how that affected the model. The base model is also given for comparison:<br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:test4.jpg|Figure 2 - Reduced Kinetic Constant for Reaction 11 and 13, One Order of Magnitude<br />
Image:test5.jpg|Figure 3 - Reduced Kinetic constant for Reaction 11 and 13, two Orders of Magnitude<br />
</gallery></center><br><br />
<br />
As can be seen the results were disappointing. ATc still out-competes everything else at high aTc concentrations it seems.<br><br />
<br />
The new models created were called '''smad_ttn_test6''' which took the above idea to the extreme. In order to see how aTc really affected the model, I decided to make sure cutting it out entirely came out with a logical graph. So reaction 11 and 13 kinetic constants were set to zero.<br><br />
<br />
The two other models were '''smad_ttn_test7''' and '''smad_ttn_test8'''. These were made to solve the problem with the non-diluting tetR2:aTc and tetR2:aTc2 complexes. In this case I eliminated tetR2:aTc and tetR2:aTc2 by changing any reaction containing those species to instead result in tetR2 and aTc. This would allow tetR2 to more accurately divide (although it would sacrifice some accuracy as well, since these complexes do in fact exist). Test8 is similar, but it also checked to see how increasing the decomposition of tetR2 (reaction 35, k=2.89E-04 1/s, increase to k=2.89E-03) would affect this modified model.<br><br />
<br />
<br />
'''Ben'''<br><br />
[[Image:Tnnnew28.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew30.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/23_July_2009Minnesota/23 July 20092009-07-30T20:11:16Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/22 July 2009|Go to Previous Day (July 22)]]'''|| width=158|'''[[Minnesota/24 July 2009|Go to Next Day (July 24)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I got back the results for test3, and also created tow programs to test whether making tetR2 less attracted to aTc would make a significant difference. I created two programs, '''smad_ttn_test4''' which looked at reducing tetR2:aTc production (the kinetic constant for reactions 11 and 13 were reduced from 1E+08 1/(Ms) to 1E+07 1/(Ms)), and '''smad_ttn_test5''' which further reduced the same constant to 1E+06 1/(Ms).<br><br />
<br />
Test3 came back and it show below along with the base for comparison. Test3 checked to see what would occur if tetR2:aTc and tetR2:aTc2 were set to Split on Division:<br><br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:test3.jpg|Figure 2 - tetR2:aTc and tetR2:aTc2 Split on Division<br />
</gallery></center><br />
<br />
As can be seen it has dramatic, but ultimately incorrect results. The results are a by-product of aTc dilution instead of tetR2 dilution as desired. I'll have to look at more ideas for how to model this correctly.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnnew24.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew26.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/22_July_2009Minnesota/22 July 20092009-07-30T20:03:55Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/21 July 2009|Go to Previous Day (July 21)]]'''|| width=158|'''[[Minnesota/23 July 2009|Go to Next Day (July 23)]]'''<br />
|}<br />
'''Patrick'''<br><br />
I created a program to explore the possibility that setting tetR2:aTc and tetR2:aTc2 complexes to "Split on Division" may help the accuracy of the model and allow for more manipulation of the shape of the graph. It is called '''smad_ttn_test3'''.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnnew20.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew22.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/21_July_2009Minnesota/21 July 20092009-07-30T20:01:09Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/20 July 2009|Go to Previous Day (July 20)]]'''|| width=158|'''[[Minnesota/22 July 2009|Go to Next Day (July 22)]]'''<br />
|}<br />
'''Patrick'''<br><br />
smad_ben did not run properly. The issue that Ben was having was eliminated, so I never reran the model.<br><br />
<br />
Today I looked at how the tetR2 concentrations are behaving as a possible reason why I am finding it difficult to shift the point at which GFP concentration reaches a steady state. The issue I think is that tetR2 splits on division, and thus is diluted if tetR2 exists in appreciable quantities in the cell. This occurs at low aTc concentrations (as noted previously in the calendar). But tetR2:aTc and tetR2:aTc2 do not split on division. This is to avoid eliminating aTc from the system. So at high aTc concentrations tetR2 is much more prevalent. This problem will be explored further in the coming days.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnnew16.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br><br />
<br><br />
[[Image:Tnnnew18.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/20_July_2009Minnesota/20 July 20092009-07-30T19:52:44Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/17 July 2009|Go to Previous Day (July 17)]]'''|| width=158|'''[[Minnesota/21 July 2009|Go to Next Day (July 21)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today, in order to help out Ben I created an equivalent model to make sure the results were consistent. I named this model '''smad_ben''', it essentially just deleted all the useless equations to make sure the models Ben were running made sense.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky35.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/17_July_2009Minnesota/17 July 20092009-07-30T19:50:22Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
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'''Patrick'''<br><br />
got back base_nsDNA, which was created to test the validity of modeling only a single tetO1 would be accurate for a high plasmid cell. This result is shown below along with the base model:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:nsDNA.jpg|Figure 2 - TTN Model with Reduced nsDNA Amount<br />
</gallery></center><br><br />
<br />
As can be seen, the additional tetO1 doesn't affect the model significantly since the amount of nsDNA is still enough to out-compete the operons in roughly the same proportion.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky33.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/17_July_2009Minnesota/17 July 20092009-07-30T19:49:36Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/16 July 2009|Go to Previous Day (July 16)]]'''|| width=158|'''[[Minnesota/20 July 2009|Go to Next Day (July 20)]]'''<br />
|}<br />
'''Patrick'''<br><br />
got back base_nsDNA, which was created to test the validity of modeling only a single tetO1 would be accurate for a high plasmid cell. This result is shown below along with the base model:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:nsDNA.jpg|Figure 2 - TTN Model with Reduced nsDNA Amount<br />
</gallery></center><br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky33.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/17_July_2009Minnesota/17 July 20092009-07-30T19:49:20Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/16 July 2009|Go to Previous Day (July 16)]]'''|| width=158|'''[[Minnesota/20 July 2009|Go to Next Day (July 20)]]'''<br />
|}<br />
'''Patrick'''<br><br />
got back base_nsDNA, which was created to test the validity of modeling only a single tetO1 would be accurate for a high plasmid cell. This result is shown below along with the base model:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:base_nsDNA.jpg|Figure 2 - TTN Model with Reduced nsDNA Amount<br />
</gallery></center><br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky33.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/16_July_2009Minnesota/16 July 20092009-07-30T19:47:16Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/15 July 2009|Go to Previous Day (July 15)]]'''|| width=158|'''[[Minnesota/17 July 2009|Go to Next Day (July 17)]]'''<br />
|}<br />
'''Patrick'''<br><br />
In order to check the validity of the assumption made yesterday, I checked one of the methods discussed. The idea that dividing the amount of nsDNA to reduce competition by 300x may lead to more accurate results.<br><br />
<br />
'''smad_ttn_base_nsDNA''' was created by dividing the initial concentration of nsDNA (500000) by 300 to give 1670.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky31.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/15_July_2009Minnesota/15 July 20092009-07-30T19:43:45Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/14 July 2009|Go to Previous Day (July 14)]]'''|| width=158|'''[[Minnesota/16 July 2009|Go to Next Day (July 16)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today there was an extended discussion about the validity of the model surrounding its condition of modeling a single tetO1 and tetO2 (and lacO1 when necessary) for each cell (it does not split on division, so each cell has one). This was done to simplify the model. Unfortunately the question arose that perhaps the model is not accurate since the cells in the experimental section are high plasmid cells (~300 plasmids). Each plasmid has 300 tetO1 and tetO2 sites then.<br><br />
<br />
Several options for correcting this were thought up. One was that since the operons are mainly in competition with non-specific DNA (nsDNA), reducing the amount of nsDNA could yield more accurate (although most likely not different) results. Another option was the increase the amount of tetO1 and tetO2 to 300. This posed several problems, most specifically the issue surrounding linking these operons into a complex such as tetO1:tetO2.<br> <br />
<br />
This method would add a significant amount of equations, Currently there are ten tetR2-tetO1 related equations, and ten tetR2-tetO2 equations. With this method there would be 10*10 = 100 equations, an increase of 80 equations. All of that for something that would more likely result in little or no improvement to the model.<br><br />
<br />
In the end it was determined the model was accurate enough for our purposes.</div>Patsmadhttp://2009.igem.org/Minnesota/14_July_2009Minnesota/14 July 20092009-07-30T19:31:57Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
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|'''[[Minnesota/13 July 2009|Go to Previous Day (July 13)]]'''|| width=158|'''[[Minnesota/15 July 2009|Go to Next Day (July 15)]]'''<br />
|}<br />
'''Patrick'''<br><br />
I got back smad_ttn_base2 from yesterday which was created to check the validity of my assumption that initial aTc concentration can be set instead of modeling the transfer of aTc from outside the cell to inside the cell. The base model is included for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1 - Base TTN Model<br />
Image:base2.jpg|Figure 2 - Base TTN Model with aTc Transfer Across the Membrane<br />
</gallery></center><br><br />
<br />
As can be seen, there is little difference between the models. Which means the two methods are equivalent<br><br />
<br />
<br><br />
[[Image:Tnnatcvariesleaky29.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/13_July_2009Minnesota/13 July 20092009-07-30T19:29:03Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/10 July 2009|Go to Previous Day (July 10)]]'''|| width=158|'''[[Minnesota/14 July 2009|Go to Next Day (July 14)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Up until now I had simply put an initial amount of aTc into the system and let the system run. This was a fairly good representation of the system all things considered. But there is an alternate way to model this: By having two aTc species. One is outside (I called it aTcO) and then a 45th reaction (aTcO --> aTc) is added to let the aTc molecules outside the cell move into the cell.<br><br />
<br />
I quickly created a new base model, '''mad_ttn_base2''', to make sure the two situations were roughly equivalent. Since it is assumed that aTc moves across the membrane more or less instantaneously, this should be the case.<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky27.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/10_July_2009Minnesota/10 July 20092009-07-30T19:19:47Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/9 July 2009|Go to Previous Day (July 9)]]'''|| width=158|'''[[Minnesota/13 July 2009|Go to Next Day (July 13)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Yesterday's results showed that lowing GFP production, while it had the potential to shift the amount of time it takes the system to reach steady state, it couldn't shift it enough even when the decomposition was set to zero. This was because cell division resulted in dilution that would make GFP reach a steady state after about 3 hours.<br><br />
<br />
The key to shifting this curve more is perhaps to change how the tetR2 molecules exist as complexes. In the TTN system tetR2 can exist by itself, as tetR2:tetO1, tetR2:aTc, tetR2:nsDNA, tetR2:tetO1:aTc, etc. about 11 different complexes. Today I focused on determining in which complexes tetR2 mostly existed as at the end of a simulation<br><br />
<br />
The results, obtained using a m-file I created, showed that at low aTc concentrations tetR2 was mostly bound to non-specific DNA (nsDNA), and floating about unbound (tetR2). At high concentrations of aTc all of it existed as tetR2:aTc2, which makes sense<br><br />
<br />
In trying to fit the experimental data, these results will be taking into consideration<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky25.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:10:06Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</gallery></center><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</gallery></center><br><br />
<br />
As can be seen the elimination of GFP production doesn't have an effect. This makes sense since source literature says GFP decomposition is minor when compared to the dilution as a result of cell division.</div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:08:49Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br><br />
<br />
As</div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:08:17Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
<\center><\gallery><br><br />
<br />
As</div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:07:54Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br><br />
<br />
As</div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:07:25Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br></div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:07:08Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><br />
<gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br></div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:06:23Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2.jpg|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br></div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:05:47Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br><br />
<br />
As can be seen, there is a small difference when the GFP was increased, but no noticeable difference when GFP decomposition was decreased. The last model set GFP decomposition to zero in order to check whether a decrease would ever significantly affect the graph:<br><br />
<br />
<center><gallery widths=400 heights=300><br />
Image:gfp_d2|Figure 4: GFP Decomposition Set to Zero<br />
</center></gallery><br></div>Patsmadhttp://2009.igem.org/Minnesota/9_July_2009Minnesota/9 July 20092009-07-30T19:03:48Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/8 July 2009|Go to Previous Day (July 8)]]'''|| width=158|'''[[Minnesota/10 July 2009|Go to Next Day (July 10)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I received and analyzed the three models created yesterday, smad_ttn_gfp_u, smad_ttn_gfp_d, smad_ttn_gfp_d2. Below are the first two models, where the kinetic constant for reaction 10 (gfp -->) was raised and lowered by an order of magnitude (from 3.21E-05 1/s). The base is shown for comparison:<br><br />
<br />
<center><gallery widths=300 heights=200><br />
Image:base.jpg|Figure 1: Base TTN Model<br />
Image:gfp_u.jpg|Figure 2: Increased GFP Decomposition Rate (3.21E-04)<br />
Image:gfp_d.jpg|Figure 3: Decreased GFP Decomposition Rate (3.21E-06)<br />
</center></gallery><br></div>Patsmadhttp://2009.igem.org/Minnesota/8_July_2009Minnesota/8 July 20092009-07-30T18:59:52Z<p>Patsmad: </p>
<hr />
<div>{|style="align:left" width="965"<br />
|-<br />
|'''[[Team:Minnesota/Modeling| Back to Notebook Home]]'''<br />
|-<br />
|'''[[Minnesota/7 July 2009|Go to Previous Day (July 7)]]'''|| width=158|'''[[Minnesota/9 July 2009|Go to Next Day (July 9)]]'''<br />
|}<br />
'''Patrick'''<br><br />
Today I looked at changing the decomposition rate of GFP to see if that affected the amount of GFP significantly.<br><br />
<br />
'''smad_ttn_gfp_u/d''' - These two models simply looked at changing the gfp decomposition (i.e. gfp -->, reaction 10) from 3.21E-05 1/s by orders of magnitude (gfp_u k=3.21E-04 1/s, gfp_d k=3.21E-06 1/s).<br><br />
<br />
'''smad_ttn_gfp_d2''' - This third model looked at eliminating gfp decomposition completely (k = 0 for reaction 10).<br><br />
<br />
'''Ben'''<br><br />
[[Image:Tnnatcvariesleaky23.jpg|480px]][[Image:Tnnexperiment.jpg|480px]]<br></div>Patsmad