http://2008.igem.org/wiki/index.php?title=Special:Contributions/Cbarcus&feed=atom&limit=50&target=Cbarcus&year=&month=2008.igem.org - User contributions [en]2024-03-29T07:18:55ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:16:27Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
{|align="justify"<br />
|[[Image:Indigo_Concentration.png|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the initial amount of Xgal in the cell (500mM). We see that as the pathway of SOS continues to function, more and more beta-gal (RecA*) gets produced and the rate of Xgal cleavage continues to go up exponentially.<br />
|-<br />
|}<br />
<br />
''Calculations were performed in MathCad, numerical data was exported to Excel and graphed for the plots above.''<br />
<br />
==Governing Rate Equations==<br />
<br />
The governing rate equations can be viewed in both the presentation and posters on the project page.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:13:47Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
{|align="justify"<br />
|[[Image:Indigo_Concentration.png|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the initial amount of Xgal in the cell (500mM). We see that as the pathway of SOS continues to function, more and more beta-gal (RecA*) gets produced and the rate of Xgal cleavage continues to go up exponentially.<br />
|-<br />
|}<br />
<br />
''Calculations were performed in MathCad, numerical data was exported to Excel and graphed for the plots above.''<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:12:41Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
{|align="justify"<br />
|[[Image:Indigo_Concentration.png|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the initial amount of Xgal in the cell (500mM). We see that as the pathway of SOS continues to function, more and more beta-gal (RecA*) gets produced and the rate of Xgal cleavage continues to go up exponentially.<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:12:12Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
{|align="justify"<br />
|[[Image:Indigo_concentration.png|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the initial amount of Xgal in the cell (500mM). We see that as the pathway of SOS continues to function, more and more beta-gal (RecA*) gets produced and the rate of Xgal cleavage continues to go up exponentially.<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:11:26Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
{|align="justify"<br />
|[[Image:indigo_concentration.png|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the initial amount of Xgal in the cell (500mM). We see that as the pathway of SOS continues to function, more and more beta-gal (RecA*) gets produced and the rate of Xgal cleavage continues to go up exponentially.<br />
|-<br />
|}<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/File:Indigo_Concentration.pngFile:Indigo Concentration.png2008-10-30T02:08:22Z<p>Cbarcus: The concentration of indigo within the cell as a function of time.</p>
<hr />
<div>The concentration of indigo within the cell as a function of time.</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-30T02:05:01Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
==Important Note on the above Beta-galactosidase models==<br />
<br />
The above models are innacurate. Upon further viewing of the model, an epiphany occurred (with a little help from Dr. Rickus). Since the concentration of the substrate (Xgal) is on the order of 2500 times more prevalent in the cell than the Km value, the reaction will proceed at Vmax. We know that Vmax is a function of Kcat and the concentration of enzyme. We make an assumption that the concentration of beta-gal molecules is equivalent to that of RecA* that was found in the SOS model. Using Avagadro's number and the fact that the dimensionless RecA* concentration can be converted to molar concentration by multiplying by the initial concentration of RecA.<br />
<br />
We can then fit this data utilizing a cubic spline and utilize it in the model of Vmax.<br />
<br />
We then use Runge-Kutta 4th order fixed step to find when the indigo concentration reaches 500 mM (the concentration of Xgal throughout the cell, assumed to be constant due to the large concentration on the plate). This happens at approximately 6 minutes after UV dosage.<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T20:13:55Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_8_5_min_coop.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds and acts cooperatively.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/File:X_gal_cleave_8_5_min_coop.jpgFile:X gal cleave 8 5 min coop.jpg2008-10-24T20:13:04Z<p>Cbarcus: The time to completely cleave the X-gal in the cell assuming a new beta-galactosidase is synthesized every 3 seconds and acts cooperatively.</p>
<hr />
<div>The time to completely cleave the X-gal in the cell assuming a new beta-galactosidase is synthesized every 3 seconds and acts cooperatively.</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T20:12:03Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
#Bachmair, A., D. Finley, et al. (1986). "INVIVO HALF-LIFE OF A PROTEIN IS A FUNCTION OF ITS AMINO-TERMINAL RESIDUE." Science 234(4773): 179-186.<br />
#Hustad, G. O., Richards.T, et al. (1973). "IMMOBILIZATION OF BETA-GALACTOSIDASE ON AN INSOLUBLE CARRIER WITH A POLYISOCYANATE POLYMER .2. KINETICS AND STABILITY." Journal of Dairy Science 56(9): 1118-1122.<br />
#Sharp, A. K., G. Kay, et al. (1969). "KINETICS OF BETA-GALACTOSIDASE ATTACHED TO POROUS CELLULOSE SHEETS." Biotechnology and Bioengineering 11(3): 363-&.<br />
#Berg, J. T., JL. Stryer, L. (2006). Biochemistry. New York, NY, W.H. Freeman and Company.<br />
<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
# The uninduced level of beta-galactosidase in the cell is 10 molecules. (Stryer, 2006).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
# The enzymes do not act cooperatively. This means that when one enzyme is formed, it does NOT speed up the reaction of the other enzymes around. This causes a large time lag. A model assuming cooperativeness is shown below.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
''All calculations were done in Microsoft Excel with basic algebraic manipulations and convolution principles by Craig Barcus.''<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T20:01:58Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T20:00:50Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_230_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.<br />
|-<br />
|}<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/File:X_gal_cleave_230_min_half_life.jpgFile:X gal cleave 230 min half life.jpg2008-10-24T20:00:20Z<p>Cbarcus: The time to completely cleave all of the X-gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.</p>
<hr />
<div>The time to completely cleave all of the X-gal in the cell when a new beta-galactosidase molecule is synthesized every 15 seconds.</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T19:58:11Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 1 second.<br />
|}<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_70_min_half_life.jpg|10px|center|frame]]<br />
This image shows the time it takes to complete cleave the X-Gal in the cell when a new beta-galactosidase molecule is synthesized every 3 seconds.<br />
|-<br />
|}<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/File:X_gal_cleave_70_min_half_life.jpgFile:X gal cleave 70 min half life.jpg2008-10-24T19:55:53Z<p>Cbarcus: The time to cleave 100% of the X-gal in the cell with a half-life of 60 minutes and a rate of formation of 3 seconds.</p>
<hr />
<div>The time to cleave 100% of the X-gal in the cell with a half-life of 60 minutes and a rate of formation of 3 seconds.</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-10-24T19:54:52Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:PUlogo.jpg|250px|center]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
|}<br />
<br />
<br />
==Modeling the cleavage of X-gal by Beta-galactosidase==<br />
<br />
'''Assumptions'''<br />
# The bacteria have been on the X-gal plate sufficiently long for the X-gal to be in equilibrium with the surface and the cell. This means that the cell has the same concentration of X-gal within its cell wall as the surface outside.<br />
# There is no diffusion limitation with this system. As soon as an enzyme is produced, it immediately can bind and cleave X-gal.<br />
# The enzymes follow a Michaelis-Menten type enzymatic reaction. Km for this system is 0.2 mM and Vm was calculated to be 4.826 M/min. (Sharp et al. 1969).<br />
# Kcat was calculated at 1.52*10^(-20) moles X-Gal / molecule beta-Gal*min. This is the rate of X-gal Cleava<br />
# The initial concentration of X-gal in the cell is: 7.48*10^(-16) moles.<br />
# Half-life of beta-gal is 60 minutes. After the half-life, the enzyme no longer functions properly and is recycled. (Bachmair et al. 1986).<br />
<br />
'''Schemes for the different models'''<br />
<br />
For the three graphs below, the following scheme was utilized in Microsoft Excel:<br />
<br />
# The enzyme will only function for 60 minutes, therefore, any complete cleavage time over 60 minutes requires that the enzymes acting for the first 60 minutes of their life will not act after that.<br />
# The cleavage of X-gal is as follows: Rate of X-gal Cleavage*number of enzyme molecules*time / Initial X-gal cleavage. This gives us a percentage which is easier to represent and analyze visually.<br />
# There are three different rates of formation of the enzyme. Data could not be found for the rate of formation, so different values were utilized.<br />
<br />
{|align="justify"<br />
|[[Image:X_gal_cleave_40_min_half_life.jpg|10px|center|frame]]<br />
|}<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/File:X_gal_cleave_40_min_half_life.jpgFile:X gal cleave 40 min half life.jpg2008-10-24T19:33:31Z<p>Cbarcus: The time to cleave 100% of the X-Gal in the cell. The beta-galactosidase has a half life of 60 minutes and one new enzyme is formed every 1 second in this model.</p>
<hr />
<div>The time to cleave 100% of the X-Gal in the cell. The beta-galactosidase has a half life of 60 minutes and one new enzyme is formed every 1 second in this model.</div>Cbarcushttp://2008.igem.org/Purdue/25_September_2008Purdue/25 September 20082008-09-25T18:52:12Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
Performed ligation reaction with LacZ and SOS:<br />
*Mixture:<br />
**1uL T4 ligase<br />
**2uL T4 buffer<br />
**5uL LacZ DNA<br />
**12uL SOS DNA<br />
*Let sit for 40 minutes at room temperature (should have been 10, but had to wait for the water bath to heat up)<br />
*Heat inactivated enzyme for 20 min at 55C<br />
<br />
*Craig, involved in learning the devil code Matlab, forgot about the enzyme. The water bath was at 58C when he took the enzyme out at 2:45PM. Heat inactivation for 1 hour, 15 minutes.<br />
<br />
'''Edited by Janie Stine'''</div>Cbarcushttp://2008.igem.org/Team:Purdue/ProjectTeam:Purdue/Project2008-09-25T18:44:24Z<p>Cbarcus: </p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
<br />
== '''Overall project''' ==<br />
This year at Purdue, our goal is to make a bacterial UV sensor for commercial application. By exploiting existing ''E. coli'' DNA repair pathways (photoreactivation and SOS); we want to eventually create a "patch" that will change colors as UV exposure increases. Thus, one would be able to test when new sunscreen needs to be applied based on actual DNA damage. Other applications could include Bacterial "tattoos" that only show up in the sun, color-changing T-shirts, etc.<br />
<br />
Biologically, we are planning to attach the ''phr'' (photoreactivation) promoter to a gene creating some kind of red color, such as RFP or prodigiosin or LacYZ on MacConkey agar. As a result, as pyrimidine dimers are formed, the natural photoreactivation pathway will be activated by the bacteria and red color will develop alongside natural DNA repair. Once more severe DNA damage occurs, the ''E. coli'' will naturally switch over to the well-documented SOS (recA) pathway. By combining the promoter for this pathway (a part used by Bangalore in 2006) with the ''lacZ'' gene, severe UV damage will make beta-galactosidase which will cleave X-gal which will create a blue pigment. Thus, our device will slowly turn red and eventually blue as the DNA damage resulting from UV radiation increases.<br />
<br />
== Project Details==<br />
Unfortunately, there is insufficient documentation regarding the photoreactivation pathway. Because this pathway is not present in humans, very little research has been done on the subject. As a result, there is no definitive source for the specific genetic code that makes up the promoter of the system. Because of this and other funding problems, the Purdue team has decided to focus on just the SOS side of the project. <br />
<br />
<br />
=== Part 1: Lit Research ===<br />
<br />
<br />
<br />
<br />
<br />
=== Part 2: Modeling ===<br />
As the Purdue team consists of mostly engineers, it is our goal to be able to mathematically model our system. A working model will help us understand the mechanisms involved in our genetic modifications, and will allow us to predict the consequences of any modifications.<br />
<br />
For more details, see the Modeling page.<br />
<br />
<br />
=== Part 3: In the Lab ===<br />
After combing the Registry of Standard Biological Parts, we found 2 parts that we could use to implement our idea. First, part J22106 (contributed by Bangalore in 2006) is the promoter for ''recA'', a central gene in the bacterial SOS pathway. Next, we found a complete ''lacZ'' (I732017) which could be attached to the promoter. Both parts are relatively OK according to the quality control tests. By cloning the sequence of promoter-reporter, we can make the traditional if-then construct often used to test promoter strength. In this case, however, we will clone it into ''lac-'' cells (so we don't get false positives). By plating on X-gal plates, those cells that have successfully transformed will turn blue.<br />
<br />
Standard Assembly methods were used. Stabs of transformed cells containing each part were obtained from iGEM. Next, a miniprep was done for each part, and each part was digested. [TO BE FINISHED]<br />
<br />
<br />
<br />
=== Part 4: Results ===<br />
No results yet! See the Modeling page for expected results...<br />
<br />
<br />
===Failed ideas???/Future Plans===<br />
*Also include ppt here?<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T16:48:57Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-|}<br />
<br />
<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T16:47:58Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-|}<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T16:47:13Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
'''Assumptions'''<br />
#The UV light intensity is constant and instantaneous.<br />
#The bacteria are not undergoing any type of DNA repair at the time of UV exposure.<br />
#Thymine dimer formation is the major DNA damage occurring.<br />
<br />
<br />
{|align="justify"<br />
|[[Image:PurdueFullModel.jpg|10px|center|frame]]<br />
|-<br />
The first figure below shows the response to a 5 J/m^2 UV irradiation. We see that the concentration of '''bound''' LexA drops considerably within the first four minutes. This also correlates with the concentration of activated RecA (RecA*) going up appreciably. After approximately 60 minutes, the concentration of RecA returns to a normal level. We therefore consider this the "stopping" point of SOS.<br />
<br />
The problems with this model include:<br />
#The model is based on an instantaneous irradiation of UV at 5 J/m^2.<br />
#The model does not account for ''continuous'' UV exposure.<br />
#The model does not account for any other proteins/genes that may be involved in SOS (ie. SulA).<br />
<br />
The benefits of this model include:<br />
#Giving us a mathematical, manipulateable model to mend for our purposes.<br />
#Showing the general trend of how SOS behaves.<br />
#Gives us a time frame for how our color reporters need to work to be feasible.<br />
<br />
<br />
|[[Image:PurdueIntersectionsModel.jpg|10px|center|frame]]<br />
|-<br />
<br />
<br />
The second figure provides us with a close-up view of the increase in RecA* and decreases of LexA and RecA. We see that an artifact occurs around the 30 second mark. This artifact is related to the step-wise function that occurs in the model. Over the larger time frame, this artifact is negligible. <br />
<br />
Since we have no quantitative data on the activation and deactivation of SOS, we must make an assumption as to when SOS truly starts to take effect. We will consider the intersection of LexA and RecA* to be the initial time when SOS can start repairing the damage accrued by UV irradiation.<br />
<br />
''All Calculations and the figures above were performed in MathCad, utilizing built in Runge-Kutta 4th order function, by Craig Barcus, utilizing the mathematical model presented by SV. Aksenov in 1997.''<br />
<br />
|}<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T16:05:30Z<p>Cbarcus: </p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
==Modeling the SOS response in a ''uvr-'' mutant (No nucleotide excision repair)==<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T15:00:40Z<p>Cbarcus: /* Modeling References */</p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T15:00:33Z<p>Cbarcus: /* Modeling References */</p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T15:00:21Z<p>Cbarcus: /* Modeling References */</p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. "Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation". [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
<br />
<br />
#2005. "Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T14:59:51Z<p>Cbarcus: /* Modeling References */</p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. Mathematical model of the SOS response regulation of an excision repair deficient mutant of ''Escherichia coli'' after UV light irradation. [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
<br />
<br />
#2005. Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ModelingTeam:Purdue/Modeling2008-09-25T14:59:32Z<p>Cbarcus: /* Modeling References */</p>
<hr />
<div>{|align="justify"<br />
|[[Image:Example_logo.png|200px|center|frame]]<br />
|-<br />
|}<br />
<br />
==Modeling Objectives==<br />
# Develop a mechanistics ODE model of the population to predict gene expression dynamics<br />
# Question? .... Will the color production be fast enough to be useful to the user? Or will it be too late?<br />
# What is the relationship between UV exposure and reporter gene expression? <br />
# Can we construct a useful calibration curve of color as a function of UV?<br />
<br />
==Modeling References==<br />
#1997. Mathematical model of the SOS response regulation of an excision repair deficient mutant of '''Escherichia coli''' after UV light irradation. [http://www2.lib.purdue.edu:2184/science?_ob=ArticleURL&_udi=B6WMD-45KKS62-5S&_user=29441&_coverDate=05%2F21%2F1997&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=29441&md5=7297869ffc31d1edfcd20856301793a5]. Off-the-shelf mechanistic model of the SOS response. Utilized as the basic model for our system.<br />
<br />
<br />
#2005. Response times and mechanisms of SOS induction by attaching promoters to GFP: "Precise Temporal Modulation in the Response of the SOS DNA Repair Network in Individual Bacteria" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601]. Potential Validation Data Set. Model should predict similar dynamics.<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/ProjectTeam:Purdue/Project2008-09-25T14:54:55Z<p>Cbarcus: /* Overall project */</p>
<hr />
<div><!--- The Mission, Experiments ---><br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
This year at Purdue, our goal is to make a bacterial UV sensor for commercial application. By exploiting existing ''E. coli'' DNA repair pathways (photoreactivation and SOS), we want to eventually create a "patch" that will change colors as UV exposure increases. Thus, one would be able to test when new sunscreen needs to be applied based on actual DNA damage. Other applications could include Bacterial "tattoos" that only show up in the sun, color-changing T-shirts, etc.<br />
<br />
Biologically, we are planning to attach the ''phr'' (photoreactivation) promoter to a gene creating some kind of red color, such as RFP or prodigiosin or LacYZ on MacConkey agar. As a result, as pyrimidine dimers are formed, the natural photoreactivation pathway will be activated by the bacteria and red color will develop alongside natural DNA repair. Once more severe DNA damage occurs, the ''E. coli'' will naturally switch over to the well-documented SOS (recA) pathway. By combining the promoter for this pathway (a part used by Bangalore in 2006) with the ''lacZ'' gene, severe UV damage will make beta-galactosidase which will cleave X-gal which will create a blue pigment. Thus, our device will slowly turn red and eventually blue as the DNA damage resulting from UV radiation increases.<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
<br />
=== Part 2 ===<br />
<br />
<br />
<br />
<br />
<br />
=== The Experiments ===<br />
<br />
<br />
<br />
<br />
=== Part 3 ===<br />
<br />
<br />
<br />
<br />
== Results ==<br />
<br />
<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Purdue/7_July_2008Purdue/7 July 20082008-07-07T19:51:50Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
==Death of the lycopene gene==<br />
<br />
Was in contact with Dr. Chris French from the University of Edinburgh over the weekend about their BioBrick part that encoded the lycopene producing gene. Dr. French stated that the part worked only marginally and that it took a long time to produce the faint pink color they recorded. Upon further contact with Dr. French, the team decided that going another route would be more beneficial to our cause in the immediate future. Further information about the carotenoid producing genes will be very useful in latter production of our UV bio-sensor.<br />
<br />
A new search was undertaken by Janie and Craig to find a new color responder. Briefly, luciferase was considered, but then neglected when the availability of luciferin was found. Craig remembered his Microbiology class and the fact that certain differential medias selected bacteria on the basis of metabolic activity. MacConkey Agar was looked up and we have decided to attempt a construct containing the lacZ alpha fragment and the lacY gene, transformed in behind the ''phr'' promoter that when activated will form pink colonies on the agar. The SOS response will remain the same with X-gal and the blue producing color. Part numbers ('''insert here''') will be incorporated to test this theory.<br />
<br />
'''Edited by Craig Barcus and Janie Stine'''<br />
<br />
==Shipment of DNA==<br />
<br />
Katie Clifford has proved instrumental in obtaining more SOS and lacZ DNA after our failed attempts at transforming ourselves. Further research into the problems of our transformations will be taking place in the near future. Tubes innoculated with glycerol stocks of the library cells were shipped out today and should arrive either tomorrow or the next day.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/2_July_2008Purdue/2 July 20082008-07-03T18:41:01Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
===IGEM Meeting===<br />
'''Members Present:'''<br />
*Craig Barcus<br />
*Janie Stine<br />
*Jessamine Osborne<br />
*Jenna Rickus<br />
*David Jaroch<br />
<br />
<br />
'''Minutes'''<br />
*Talked about plans for transformation tomorrow<br />
*Discussed fund-raising<br />
**Edited draft of letter to companies<br />
**Possible people to target<br />
**Perks for sponsors<br />
*Picked a logo!<br />
<br />
<br />
'''Edited by Janie Stine'''</div>Cbarcushttp://2008.igem.org/Purdue/30_June_2008Purdue/30 June 20082008-06-30T12:43:45Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
===Problems with Bacterial Transformations===<br />
<br />
The experiments done on Friday, June 27 all turned out '''negative''' on the Amp plates, except for the previously transfected GFP labeled bacteria from Dr. Clase. The GFP ''E. coli'' grew extremely well. This proves that it is not the Amp agar causing the problem<br />
<br />
In the four tubes innoculated, all four show signs of bacterial growth, even the one that does not theoretically have any bacteria growing in it. This means that either the tips we are using are contaminated (will use freshly autoclaven tips next time), the innoculation tubes are contaminated (will use freshly autoclaven tubes next time), or the media is contaminated (will set out 10 mL at 37C during the day today. Check at end of day.)<br />
<br />
Due to these results, I feel it is prudent to conclude that our transformations failed miserably and the cause is due to overcompetition from wild type species accidentally included in the culturing medium for the overnight recovery. As for the one-hour recovery, I feel that there was simply not enough time to recover from the heat shock and possibly that there was not enough DNA to fully transform the cells.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/27_June_2008Purdue/27 June 20082008-06-27T21:05:48Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
The "transformed" cells still did not grow on an Amp plate, meaning no cells took up the LacZ or the SOS DNA. The problem is most likely the DNA and/or the DNA prepping procedure. We'll need to contact our iGEM contact, and ask what to do.<br />
<br />
Later today, we'll inoculate another liquid media tube (so as not to lose the stock of cells), and plate again on newer Amp plates (to see if the old agar was too old or something).<br />
<br />
'''Edited by Janie Stine'''<br />
<br />
Innoculated three new tubes, one with SOS, LacZ, and the GFP. Also "innoculated" as sterile tube from the stock to check for native contamination that could be taking over against our transformed bacteria. Also, took from same tubes I did the tube innoculation, I plated each bacterial strain on AMP plates. Will check Sunday morning to see if they worked.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/25_June_2008Purdue/25 June 20082008-06-25T19:16:59Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
==Second Attempt at Transformations==<br />
<br />
Obtained a new set of Chemically Compotent Cells from Larisa. Cells are considered Max Efficiency.<br />
<br />
==UV Vis Spectroscopy Data from Plasmid==<br />
<br />
Utilized UV Vis in Room 234 to get an absorbance reading from DNA plasmid to determine the amount of DNA on a single punch from the part kit.<br />
<br />
Analyzation revealed approximately 112 ug/mL, which translates to 1 ug per spot (or punch). The second transformation above used two spots of DNA.</div>Cbarcushttp://2008.igem.org/Team:Purdue/NotebookTeam:Purdue/Notebook2008-06-25T19:05:27Z<p>Cbarcus: </p>
<hr />
<div>==Notebook Calendar==<br />
{|align="justify"<br />
|width="200pt"| {{#calendar: title=Purdue |year=2008 | month=05}} <br />
|width="200pt"| {{#calendar: title=Purdue |year=2008 | month=06}} <br />
|width="200pt"| {{#calendar: title=Purdue |year=2008 | month=07}} <br />
|width="250pt"|[[Image:Purdue-logo.jpg|200px|center]]<br />
|-<br />
| {{#calendar: title=Purdue |year=2008 | month=08}}<br />
| {{#calendar: title=Purdue |year=2008 | month=09}}<br />
| {{#calendar: title=Purdue |year=2008 | month=10}}<br />
|}<br />
<br />
{|align="center"<br />
<br />
==Summary of Key Notebook Pages==<br />
'''June 2, 4''' Craig's Milk pH Data<br />
<br />
'''June 10''' Second run and results of Milk pH data.<br />
<br />
'''June 11''' Group meeting and summary of UV idea<br />
<br />
'''June 13''' List of parts to use<br />
<br />
'''June 23''' First attempt at transformation<br />
<br />
'''June 25''' Second attempt at transformation<br />
|<br />
|<br />
|[[Image:Team.png|right|frame|Your team picture]]<br />
<br />
|}<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Purdue/25_June_2008Purdue/25 June 20082008-06-25T19:04:47Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
==Second Attempt at Transformations==<br />
<br />
Obtained a new set of Chemically Compotent Cells from Larisa. Cells are considered Max Efficiency.<br />
<br />
==UV Vis Spectroscopy Data from Plasmid==<br />
<br />
Utilized UV Vis in Room 234 to get an absorbance reading from DNA plasmid to determine the amount of DNA on a single punch from the part kit.</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-24T19:21:59Z<p>Cbarcus: </p>
<hr />
<div>===VERY IMPORTANT INFORMATION ABOUT ACCESSING REFERENCES===<br />
<br />
When trying to access the research articles, most of them are through Web of Science, which means you '''must log into WoS through the Purdue Library Site''' listed below. The link is the first under Useful Tools Below. Go to the "Find a database" and click Web of Science (or PubMed depending on the article). Log in from there and then you should be able to click and retrieve the article or how to get to the article.<br />
<br />
<br />
==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
# Review article 1. [http://www2.lib.purdue.edu:2164/content/7l83vj6244wq8164/fulltext.pdf]<br />
# "Crystal Structure of thermostable DNA photolyase: pyrimidine dimer recognition mechanism." [http://www2.lib.purdue.edu:2111/sici?sici=0027-8424%282001%2998%3A24%3C13560%3ACSOTDP%3E2.0.CO%3B2-B&origin=ISI&cookieSet=1]<br />
# Review article 2. "Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase". [http://www2.lib.purdue.edu:6624/purdue?&url_ver=Z39.88-2004&url_ctx_fmt=info:ofi/fmt:kev:mtx:ctx&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.atitle=Light-driven+enzymatic+catalysis+of+DNA+repair%3A+a+review+of+recent+biophysical+studies+on+photolyase&rft.auinit=S&rft.aulast=Weber&rft.date=2005&rft.epage=23&rft.genre=article&rft.issn=0005-2728&rft.issue=1&rft.spage=1&rft.stitle=BBA-BIOENERGETICS&rft.title=BIOCHIMICA+ET+BIOPHYSICA+ACTA-BIOENERGETICS&rft.volume=1707&rfr_id=info:sid/www.isinet.com:WoK:WOS&rft_id=info:doi/10.1016%2Fj.bbabio.2004.02.010]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. <br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-24T19:19:48Z<p>Cbarcus: </p>
<hr />
<div>===VERY IMPORTANT INFORMATION ABOUT ACCESSING REFERENCES===<br />
<br />
When trying to access the research articles, most of them are through Web of Science, which means you '''must log into WoS through the Purdue Library Site''' listed below.<br />
<br />
<br />
==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
# Review article 1. [http://www2.lib.purdue.edu:2164/content/7l83vj6244wq8164/fulltext.pdf]<br />
# "Crystal Structure of thermostable DNA photolyase: pyrimidine dimer recognition mechanism." [http://www2.lib.purdue.edu:2111/sici?sici=0027-8424%282001%2998%3A24%3C13560%3ACSOTDP%3E2.0.CO%3B2-B&origin=ISI&cookieSet=1]<br />
# Review article 2. "Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase". [http://www2.lib.purdue.edu:6624/purdue?&url_ver=Z39.88-2004&url_ctx_fmt=info:ofi/fmt:kev:mtx:ctx&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.atitle=Light-driven+enzymatic+catalysis+of+DNA+repair%3A+a+review+of+recent+biophysical+studies+on+photolyase&rft.auinit=S&rft.aulast=Weber&rft.date=2005&rft.epage=23&rft.genre=article&rft.issn=0005-2728&rft.issue=1&rft.spage=1&rft.stitle=BBA-BIOENERGETICS&rft.title=BIOCHIMICA+ET+BIOPHYSICA+ACTA-BIOENERGETICS&rft.volume=1707&rfr_id=info:sid/www.isinet.com:WoK:WOS&rft_id=info:doi/10.1016%2Fj.bbabio.2004.02.010]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Purdue/23_June_2008Purdue/23 June 20082008-06-23T20:02:38Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
Looked up QC information for the SOS and LacZ parts (J22106 and I732005). SOS is OK and consistent, but I732005 is Inconsistent and BAD for all analyses. <br />
<br />
Found new part that is more consistent and OK:<br />
*I732017: LacZ (full-length) + RBS in front<br />
*Location: Plate 1009, Well 11H<br />
*Plasmid: pSB1A2 (AmpR)<br />
*RBS efficiency = 1.0 (based on Elowitz 1999 Repressilator)<br />
<br />
'''Edited by Janie Stine'''<br />
<br />
<br />
===Transformation of LacZ and SOS parts===<br />
*Used OneShot MAX Efficiency DH5alpha-T1 cells (Invitrogen) and used a combo of their protocol and the iGEM protocol<br />
*Thawed on ice<br />
*Cut out parts from filter paper, warmed in 5 uL of TE 20 min.<br />
**LacZ/RBS (I732015), plate 1009, well 11H, plasmid pSB1A2<br />
**SOS promoter (J22106), plate 1003, well 12F, plasmid pSB1A2<br />
*Added all TE/DNA (~5uL) to 25uL of cells for each part<br />
*Let sit on ice 30 min. in 2 mL Eppendorfs<br />
*Put cells in 42C water bath EXACTLY 30s (no mixing or shaking)<br />
*Place on ice<br />
*Add 250uL warmed SOC to each vial<br />
*Shake EXACTLY 1 hour at 300rpm and 37C<br />
*Spread 200uL from ea. vial onto LB plates (store remainder at 4C)<br />
*Invert plates and let incubate at 37C until we leave (to get a kick-start to their growth)<br />
*Let incubate at room temperature overnight (We can't get to the cells for more than 14 hours, so we're forcing them to grow more slowly)<br />
<br />
<br />
'''Edited by Janie Stine'''<br />
<br />
===Sol-Gel Vapor Spray===<br />
<br />
Fluorescence checked with confocal microscope with Jennie. <br />
<br />
One plate that was visibly confounded has fluorescence that ranged over all red/green/blue ranges. Most likely silica that has clumped and refracted light.<br />
<br />
One sprayed plate showed few bacteria that fluoresced green, confirming our suspicion that the bacteria are encapsulated. Great result for future work.<br />
<br />
The sol-gel native (sol-gel pipetted into the plate) showed approximately the same as the spray. Good for a control of sol-gel.<br />
<br />
Janie and Jennie both looked at the fluorescence. (Craig is color blind and could not distinguish.) Both concurred that what was seen in the two positive plates were what was expected of GFP bacteria.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-20T18:29:20Z<p>Cbarcus: </p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
# Review article 1. [http://www2.lib.purdue.edu:2164/content/7l83vj6244wq8164/fulltext.pdf]<br />
# "Crystal Structure of thermostable DNA photolyase: pyrimidine dimer recognition mechanism." [http://www2.lib.purdue.edu:2111/sici?sici=0027-8424%282001%2998%3A24%3C13560%3ACSOTDP%3E2.0.CO%3B2-B&origin=ISI&cookieSet=1]<br />
# Review article 2. "Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase". [http://www2.lib.purdue.edu:6624/purdue?&url_ver=Z39.88-2004&url_ctx_fmt=info:ofi/fmt:kev:mtx:ctx&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.atitle=Light-driven+enzymatic+catalysis+of+DNA+repair%3A+a+review+of+recent+biophysical+studies+on+photolyase&rft.auinit=S&rft.aulast=Weber&rft.date=2005&rft.epage=23&rft.genre=article&rft.issn=0005-2728&rft.issue=1&rft.spage=1&rft.stitle=BBA-BIOENERGETICS&rft.title=BIOCHIMICA+ET+BIOPHYSICA+ACTA-BIOENERGETICS&rft.volume=1707&rfr_id=info:sid/www.isinet.com:WoK:WOS&rft_id=info:doi/10.1016%2Fj.bbabio.2004.02.010]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-20T18:28:18Z<p>Cbarcus: </p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
# Review article 1. [http://www2.lib.purdue.edu:2164/content/7l83vj6244wq8164/fulltext.pdf]<br />
# "Crystal Structure of thermostable DNA photolyase: pyrimidine dimer recognition mechanism." [http://www2.lib.purdue.edu:2111/sici?sici=0027-8424%282001%2998%3A24%3C13560%3ACSOTDP%3E2.0.CO%3B2-B&origin=ISI&cookieSet=1]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-20T17:26:53Z<p>Cbarcus: </p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
# Review article 1. [http://www2.lib.purdue.edu:2164/content/7l83vj6244wq8164/fulltext.pdf]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-20T17:20:21Z<p>Cbarcus: </p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
# 1987 "Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/2823069?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
#1987 "Induction of phr gene expression by pyrimidine dimers in Escherichia coli." [http://www.ncbi.nlm.nih.gov/pubmed/3313446?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# 1989 "The LexA protein does not bind specifically to the two SOS box-like sequences immediately 5' to the phr gene." [http://www.ncbi.nlm.nih.gov/pubmed/2509902?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]<br />
# 1995 "Promoters of the phr gene in E. coli K-12": Ma, Chuping and Claud S. Rupert. ''Molecular and General Genetics''. v.248 Pgs. 52-58. 1995.<br />
# 2001 "Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation." [http://www.ncbi.nlm.nih.gov/pubmed/11470570?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]<br />
# "Detecting UV damage in single DNA molecules by AFM" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=CombineSearches&qid=3&SID=1A8JLc54jOBnIioE2C3&page=1&doc=6]<br />
<br />
===UV Basics and Skin Cancer Statistics===<br />
#UV varies on any given day. What is the UV index? EPA definition and UV map predictions for US [http://www.epa.gov/sunwise/uvindex.html]<br />
#Skin Cancer Stats from the American Academy of Dermatology [http://www.aad.org/media/background/factsheets/fact_skincancer.html]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Purdue/13_June_2008Purdue/13 June 20082008-06-13T17:59:13Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
#Possible colors include napthalene, indigo, lycopene.<br />
#Research article by Murdock, Ensley, Serdar, and Thalen: ''Construction of Metabolic Operons Catalyzing the Biosynthesis of Indigo.'' [http://www2.lib.purdue.edu:2149/summary.do?product=WOS&doc=1&qid=10&SID=4BEdipcCm1A35k4CKg6&search_mode=AdvancedSearch]<br />
<br />
Bacterial study started on the 10th failed (plate dried out).<br />
<br />
New test started. GFP ''E. coli'' pulled from -80C and two LB tubes innoculated. Put in 37C shaker at 220 rpm over the weekend.<br />
<br />
'''Edited by Craig Barcus'''<br />
<br />
<br />
== Research on Genes and Plasmids ==<br />
<br />
#Found two different parts on the registry '''Part:BBa_I742120''', a lycopene producing gene for red color, and '''Part:BBa_I742144''' for an blue producing colony.<br />
#Janie and I discussed the possibility of having a triple color entendre. When no UV irridation is occurring, the patch will be clear. When minor damage occurs that causes the activation of the ''phr'' light repair mechanism, the bacteria can produce the lycopene (or sam8). <br />
#When the UV damage becomes too substantial, and the SOS pathway becomes activated, the other color will be produced, giving a warning sign that might as well read "'''You are extra-crispy, get out of the sun now!!'''". <br />
#With the sam8 gene (blue colony), a dependence on lactose seems to occur. This would fit right into our plans, allowing the gene to work with a double safety net. It will only produce when both lactose and UV damage has occurred.<br />
<br />
Possibilities of double engineering the bacteria remain to be discussed. Talking to Drs. Clase and/or Applegate about this needs to happen.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/13_June_2008Purdue/13 June 20082008-06-13T17:58:31Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
#Possible colors include napthalene, indigo, lycopene.<br />
#Research article by Murdock, Ensley, Serdar, and Thalen: ''Construction of Metabolic Operons Catalyzing the Biosynthesis of Indigo.'' [http://www2.lib.purdue.edu:2149/summary.do?product=WOS&doc=1&qid=10&SID=4BEdipcCm1A35k4CKg6&search_mode=AdvancedSearch]<br />
<br />
Bacterial study started on the 10th failed (plate dried out).<br />
<br />
New test started. GFP ''E. coli'' pulled from -80C and two LB tubes innoculated. Put in 37C shaker at 220 rpm over the weekend.<br />
<br />
'''Edited by Craig Barcus'''<br />
<br />
<br />
== Research on Genes and Plasmids ==<br />
<br />
#Found two different parts on the registry [[Part:BBa_I742120]], a lycopene producing gene for red color, and [[Part:BBa_I742144]] for an blue producing colony.<br />
#Janie and I discussed the possibility of having a triple color entendre. When no UV irridation is occurring, the patch will be clear. When minor damage occurs that causes the activation of the ''phr'' light repair mechanism, the bacteria can produce the lycopene (or sam8). <br />
#When the UV damage becomes too substantial, and the SOS pathway becomes activated, the other color will be produced, giving a warning sign that might as well read "'''You are extra-crispy, get out of the sun now!!'''". <br />
#With the sam8 gene (blue colony), a dependence on lactose seems to occur. This would fit right into our plans, allowing the gene to work with a double safety net. It will only produce when both lactose and UV damage has occurred.<br />
<br />
Possibilities of double engineering the bacteria remain to be discussed. Talking to Drs. Clase and/or Applegate about this needs to happen.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/13_June_2008Purdue/13 June 20082008-06-13T17:48:26Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
#Possible colors include napthalene, indigo, lycopene.<br />
#Research article by Murdock, Ensley, Serdar, and Thalen: ''Construction of Metabolic Operons Catalyzing the Biosynthesis of Indigo.'' [http://www2.lib.purdue.edu:2149/summary.do?product=WOS&doc=1&qid=10&SID=4BEdipcCm1A35k4CKg6&search_mode=AdvancedSearch]<br />
<br />
Bacterial study started on the 10th failed (plate dried out).<br />
<br />
New test started. GFP ''E. coli'' pulled from -80C and two LB tubes innoculated. Put in 37C shaker at 220 rpm over the weekend.<br />
<br />
'''Edited by Craig Barcus'''</div>Cbarcushttp://2008.igem.org/Purdue/13_June_2008Purdue/13 June 20082008-06-13T17:48:08Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
#Possible colors include napthalene, indigo, lycopene.<br />
#Research article by Murdock, Ensley, Serdar, and Thalen: ''Construction of Metabolic Operons Catalyzing the Biosynthesis of Indigo.'' [http://www2.lib.purdue.edu:2149/summary.do?product=WOS&doc=1&qid=10&SID=4BEdipcCm1A35k4CKg6&search_mode=AdvancedSearch]<br />
<br />
Bacterial study started on the 10th failed (plate dried out).<br />
<br />
New test started. GFP ''E. coli'' pulled from -80C and two LB tubes innoculated. Put in 37C shaker at 220 rpm over the weekend.</div>Cbarcushttp://2008.igem.org/Purdue/13_June_2008Purdue/13 June 20082008-06-13T14:00:22Z<p>Cbarcus: </p>
<hr />
<div>[[Team:Purdue/Notebook | Click Here to return to the notebook.]]<br />
<br />
#Possible colors include napthalene, indigo, lycopene.<br />
#Research article by Murdock, Ensley, Serdar, and Thalen: ''Construction of Metabolic Operons Catalyzing the Biosynthesis of Indigo.'' [http://www2.lib.purdue.edu:2149/summary.do?product=WOS&doc=1&qid=10&SID=4BEdipcCm1A35k4CKg6&search_mode=AdvancedSearch]</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-12T18:20:36Z<p>Cbarcus: /* UV Research */</p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
#Sequences of the ''E. coli'' Photolyase gene and protein: [http://www2.lib.purdue.edu:2479/cgi/reprint/259/9/6033]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/Tools_and_ReferencesTeam:Purdue/Tools and References2008-06-12T17:56:57Z<p>Cbarcus: /* UV Research */</p>
<hr />
<div>==Useful References==<br />
===Milk Research===<br />
#A general '''overview of milk''' and how factors affect its lifespan and production, [http://www.ilri.org/InfoServ/Webpub/Fulldocs/ILCA_Manual4/Toc.htm#TopOfPage].'''<br />
#University of Guelph '''Overview of Milk''': [http://www.foodsci.uoguelph.ca/dairyedu/home.html].<br />
#Investigations into the '''activity of enzymes produced by spoilage-causing bacteria''': a possible basis for improved shelf-life estimation: Braun et al. ''Food Microbiology''. v.16 Pgs. 531-540. 1999.<br />
#'''Microbial and biochemical spoilage of foods: An overview.''' intVeld, JHJH. ''International Journal of Food Microbiology''. v.33 Pgs. 1-18. 1996.<br />
#'''pH & gene expression''' old review (1993), but useful. [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2958.1993.tb01198.x?cookieSet=1]<br />
#Effect of Temperature, Pressure, and CO2 Concentration on the pH of milk: Ma et al. ''Journal of Dairy Science''. v.86 Pgs. 3822-3830. 2003. [http://jds.fass.org/cgi/content/full/86/12/3822]<br />
#''Dairy Science and Technology''. 2nd ed: Walstra et al. 2006 CRC Press.<br />
<br />
===UV Research===<br />
#An overview of the SOS genes, [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1174825].<br />
#A related article, examining response times and mechanisms of SOS induction by attaching promoters to GFP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1151601].<br />
#Web of Science page for "Binding of Photolyase of ''E. coli'' to UV-Damaged DNA" [http://www2.lib.purdue.edu:2149/full_record.do?product=WOS&search_mode=AdvancedSearch&qid=8&SID=3F4ocg8a@Agl43ijgDe&page=35&doc=344]<br />
<br />
==Useful Tools==<br />
<br />
#Go through Purdue Libraries [http://www.lib.purdue.edu/] to get to the research databases. I prefer Web of Science.<br />
#Wiley Protocols in Molecular Biology. Link will only work on Purdue Recognized Computers. [http://www.mrw.interscience.wiley.com/emrw/9780471142720/cp/cpmb/toc]<br />
#Biobricks Parts Registry [http://partsregistry.org/Main_Page]<br />
<br />
<br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}</div>Cbarcushttp://2008.igem.org/Team:Purdue/NotebookTeam:Purdue/Notebook2008-06-10T19:46:22Z<p>Cbarcus: /* Summary of Key Notebook Pages */</p>
<hr />
<div>{{#calendar: title=Purdue |year=2008 | month=05}} {{#calendar: title=Purdue |year=2008 | month=06}} {{#calendar: title=Purdue |year=2008 | month=07}} <br />
<br />
<br />
{|align="justify"<br />
|<br />
<br />
==Summary of Key Notebook Pages==<br />
'''June 2, 4''' Craig's Milk pH Data<br />
<br />
'''June 10''' Second run and results of Milk pH data.<br />
<br />
|[[Image:Purdue-logo.jpg|200px|right|frame]]<br />
|-<br />
|<br />
|[[Image:Team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
<br />
|align="center"|[[Team:Purdue | Team Example 2]]<br />
|}<br />
<!--- The Mission, Experiments ---><br />
<br />
{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:Purdue|Home]]<br />
!align="center"|[[Team:Purdue/Team|The Team]]<br />
!align="center"|[[Team:Purdue/Project|The Project]]<br />
!align="center"|[[Team:Purdue/Parts|Parts Submitted to the Registry]]<br />
!align="center"|[[Team:Purdue/Modeling|Modeling]]<br />
!align="center"|[[Team:Purdue/Notebook|Notebook]]<br />
!align="center"|[[Team:Purdue/Tools and References|Tools and References]]<br />
|}<br />
<br />
==Notebook==<br />
<br />
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. <br />
<br />
Find more information on how to use the calendar feature by going to the [[Help:Calendar | general calendar page]].</div>Cbarcus