BME494s2013 Project Team1: Difference between revisions
No edit summary |
|||
Line 81: | Line 81: | ||
Julia | Julia | ||
===Our | ===Our gene switch=== | ||
==== | ====Parts==== | ||
<tab>pSB1A3-1 is a high copy number plasmid. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). The terminators bracketing pSB1A3 MCS are designed to prevent transcription from inside the MCS from reading out into the vector. | <tab>pSB1A3-1 is a high copy number plasmid. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). The terminators bracketing pSB1A3 MCS are designed to prevent transcription from inside the MCS from reading out into the vector. | ||
Line 98: | Line 98: | ||
<!-- These are lines breaks for spacing purposes. You can add or delete these as needed --> | <!-- These are lines breaks for spacing purposes. You can add or delete these as needed --> | ||
==Building: Assembly | ==Building: Assembly scheme== | ||
<!-- Illustrate and describe how you will build your lac switch. Incorporate information from Group Presentation 2 --> | <!-- Illustrate and describe how you will build your lac switch. Incorporate information from Group Presentation 2 --> | ||
Line 112: | Line 112: | ||
<!-- These are lines breaks for spacing purposes. You can add or delete these as needed --> | <!-- These are lines breaks for spacing purposes. You can add or delete these as needed --> | ||
==Testing: Modeling and GFP | ==Testing: Modeling and GFP imaging== | ||
<br> | <br> | ||
[[Image:CirEmblemHandwriteLogo2.png|thumb|140px||left|Network | [[Image:CirEmblemHandwriteLogo2.png|thumb|140px||left|Network diagram illustration of the ''lac'' model (Julia)]] | ||
===A | ===A ''lac'' switch model=== | ||
We used a previously published synthetic switch, developed by Ceroni et al.<cite>Ceroni-2010</cite>, to understand how our system could potentially be modeled and simulated. The graphic to the left depicts the relationships between the parameters of the | We used a previously published synthetic switch, developed by Ceroni et al.<cite>Ceroni-2010</cite>, to understand how our system could potentially be modeled and simulated. The graphic to the left depicts the relationships between the parameters of the ''lac'' operon switch described by Ceroni using a network diagram illustration. The parameters shown in the illustration relate to cell processes and could be used in forming a cohesive mathematical model of the cell's operation. | ||
In order to approximate the behavior of this set-up, a mathematical model can be developed based upon the relationships between the processes found in the cell. These relationships can be expressed in mathematical terms using numbers that relate to the system, including creation or decay rates, concentrations, or various constants. The actual values for these parameters can be sourced from experimentation, literature, or a predefined steady-state. | In order to approximate the behavior of this set-up, a mathematical model can be developed based upon the relationships between the processes found in the cell. These relationships can be expressed in mathematical terms using numbers that relate to the system, including creation or decay rates, concentrations, or various constants. The actual values for these parameters can be sourced from experimentation, literature, or a predefined steady-state. | ||
Line 127: | Line 127: | ||
The formula takes the concentration of the GFP protein in molecules per cell ('''"G"''') and multiplies it by the protein degradation rate in minutes<sup>-1</sup> ('''"λ<sub>G/L</sub>"'''). This results in a decay value for GPF in molecules per minute per cell. | The formula takes the concentration of the GFP protein in molecules per cell ('''"G"''') and multiplies it by the protein degradation rate in minutes<sup>-1</sup> ('''"λ<sub>G/L</sub>"'''). This results in a decay value for GPF in molecules per minute per cell. | ||
The Ceroni et al. model and the network diagram illustration use the table of variables and parameters seen below in their representation of the | The Ceroni et al. model and the network diagram illustration use the table of variables and parameters seen below in their representation of the ''lac'' switch. The variables related to a particular cell process are located near to that process in the network diagram illustration. | ||
{| border="1" class="wikitable" | {| border="1" class="wikitable" | ||
|+ '''Lac | |+ '''''Lac'' switch model: Important variables and parameters'''<cite>Ceroni-2010</cite> | ||
! Variable | ! Variable | ||
! Description | ! Description | ||
Line 233: | Line 233: | ||
[[Image:Bgal0.25.jpg|thumb|right|210px|Figure 2: Bgal Concentration vs. Time with I = 0.25]] | [[Image:Bgal0.25.jpg|thumb|right|210px|Figure 2: Bgal Concentration vs. Time with I = 0.25]] | ||
[[Image:Bgal0.064.jpg|thumb|right|210px|Figure 3: Bgal Concentration vs. Time with I = 0.064]] | [[Image:Bgal0.064.jpg|thumb|right|210px|Figure 3: Bgal Concentration vs. Time with I = 0.064]] | ||
=== | ===An interactive model=== | ||
We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system. By changing the initial concentration of input (IPTG in this case), we were able to estimate the threshold that produces an "on" state in the system. | We used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system. By changing the initial concentration of input (IPTG in this case), we were able to estimate the threshold that produces an "on" state in the system. | ||
Line 241: | Line 241: | ||
=== | ===Collecting imperical values to improve the model=== | ||
Sarah | Sarah | ||
<br> | <br> | ||
Line 263: | Line 263: | ||
==Stakeholder Assessment== | ==Stakeholder Assessment== | ||
<br> | <br> | ||
[[Image:Stakeholder-matrix.png|thumb| | [[Image:Stakeholder-matrix.png|thumb|210px|right|'''Stakeholder Matrix''']] | ||
'''SUPPORTS & UNDERSTANDS'''<br> | '''SUPPORTS & UNDERSTANDS'''<br> |
Revision as of 14:46, 28 April 2013
Home People Course Projects Course Materials Schedule Photos Wiki Editing Help
Overview & PurposeSarah
Background
"Only LacZ and LacY appear to be necessary for lactose catabolism" [3].
Design: Our genetic circuitJulia Our gene switchParts<tab>pSB1A3-1 is a high copy number plasmid. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). The terminators bracketing pSB1A3 MCS are designed to prevent transcription from inside the MCS from reading out into the vector.
Building: Assembly schemeEmily
Testing: Modeling and GFP imaging
A lac switch modelWe used a previously published synthetic switch, developed by Ceroni et al.[5], to understand how our system could potentially be modeled and simulated. The graphic to the left depicts the relationships between the parameters of the lac operon switch described by Ceroni using a network diagram illustration. The parameters shown in the illustration relate to cell processes and could be used in forming a cohesive mathematical model of the cell's operation. In order to approximate the behavior of this set-up, a mathematical model can be developed based upon the relationships between the processes found in the cell. These relationships can be expressed in mathematical terms using numbers that relate to the system, including creation or decay rates, concentrations, or various constants. The actual values for these parameters can be sourced from experimentation, literature, or a predefined steady-state. If a model is well-defined and the necessary parameters known, a person may use the model to ascertain the state of a cell at a given point in time. For example, if an experimenter wanted to know the decay of the GFP protein molecules at a given point in time in a single cell, the following equation could be written using the notation found in the table below. Decay = G × λG/L The formula takes the concentration of the GFP protein in molecules per cell ("G") and multiplies it by the protein degradation rate in minutes-1 ("λG/L"). This results in a decay value for GPF in molecules per minute per cell. The Ceroni et al. model and the network diagram illustration use the table of variables and parameters seen below in their representation of the lac switch. The variables related to a particular cell process are located near to that process in the network diagram illustration.
An interactive modelWe used a model of the natural Lac operon to understand how changing the parameter values changes the behavior of the system. By changing the initial concentration of input (IPTG in this case), we were able to estimate the threshold that produces an "on" state in the system. Initially, the code had the concentration at 0.32 which is seen in the β-galactoside (Bgal concentration) vs. time plot (Figure 1). This value was changed again to 0.25 in determining the threshold that produces this "on" state (Figure 2). After proceeding to go up and down with these a values, a threshold was indeed found where the concentration of IPTG is about 0.064 (Figure 3).
Collecting imperical values to improve the modelSarah
Stakeholder Assessment
SUPPORTS & UNDERSTANDS
Our Team
Works Cited |