Tristable/Testing Constructs

From 2007.igem.org

(Difference between revisions)
Line 110: Line 110:
We decided to to test for three  values.  The combined transcription/translation rate of each repressor, the cooperativity of each repressor and the concentration of ligand needed to deactivate each repressor.  We managed to design three tests all using the same constructs so as to minimize ligations.  These tests should determine our variables independantly, i.e. changing synthesis rate should not change cooperativity of repression.
We decided to to test for three  values.  The combined transcription/translation rate of each repressor, the cooperativity of each repressor and the concentration of ligand needed to deactivate each repressor.  We managed to design three tests all using the same constructs so as to minimize ligations.  These tests should determine our variables independantly, i.e. changing synthesis rate should not change cooperativity of repression.
-
==Synthesis Rate==
+
 
 +
===Synthesis Rate===
The combined transcription/translation rate of the repressor is the combined strength of the promoter and the RBS.  In [[Image:alphaTest.png|thumb|left|The Alpha test Architecture]]our model this is the alpha value.  Our model predicts that the alpha values for each repressor should be fairly comparable (the stable region is along the 1 to 1 to 1 line in 3D).  Since we can't change the promoter strength very easily, we will change the RBS strength to obtain similar alpha values for all repressors.
The combined transcription/translation rate of the repressor is the combined strength of the promoter and the RBS.  In [[Image:alphaTest.png|thumb|left|The Alpha test Architecture]]our model this is the alpha value.  Our model predicts that the alpha values for each repressor should be fairly comparable (the stable region is along the 1 to 1 to 1 line in 3D).  Since we can't change the promoter strength very easily, we will change the RBS strength to obtain similar alpha values for all repressors.
Line 123: Line 124:
-
==Cooperativity of Repression==
+
 
 +
 
 +
===Cooperativity of Repression===
The cooperativity describes an inherent characteristic of a repressor's repression.  In our system we want to know how [[Image:betaTest.png|thumb|left|The Beta test Architecture]]much increased repressor concentration will increase repression.  In our model, cooperativity is an exponent, beta, so that the repressor concentration is raised to the beta ([repressor]^beta = total repression).  For beta = 1, repression increases linearly with repressor concentration.  With twice as much repressor there is twice the repression.  For beta = 2, repression increases with the square of the concetration.  Twce as much repressor leads to four times the repression.  Our model predicts that our system will be more robust with greater alpha values and the tri stable region (in the graph) will be larger.  Beta must be larger than one for the system to be stable.   
The cooperativity describes an inherent characteristic of a repressor's repression.  In our system we want to know how [[Image:betaTest.png|thumb|left|The Beta test Architecture]]much increased repressor concentration will increase repression.  In our model, cooperativity is an exponent, beta, so that the repressor concentration is raised to the beta ([repressor]^beta = total repression).  For beta = 1, repression increases linearly with repressor concentration.  With twice as much repressor there is twice the repression.  For beta = 2, repression increases with the square of the concetration.  Twce as much repressor leads to four times the repression.  Our model predicts that our system will be more robust with greater alpha values and the tri stable region (in the graph) will be larger.  Beta must be larger than one for the system to be stable.   
Line 130: Line 133:
-
==Ligand Concentration==
+
 
 +
 
 +
===Ligand Concentration===
Naturally, we don't want to add more ligand than we need.  In what ever application the project might find, if we wanted to change the state again by adding a different [[Image:ligandTest.png|thumb|left|The Ligand test Architecture]]ligand, we wouldn't want excessive amounts of the first ligand floating around.
Naturally, we don't want to add more ligand than we need.  In what ever application the project might find, if we wanted to change the state again by adding a different [[Image:ligandTest.png|thumb|left|The Ligand test Architecture]]ligand, we wouldn't want excessive amounts of the first ligand floating around.

Revision as of 00:06, 25 October 2007

Brown University
Home Lead Sensor Tristable Switch Community Supplemental About us


Contents

Testing Constructs

There are two methods we could follow in designing the Switch. We could randomly try different RBSs, hope it works and if not try again without having much of an understanding of why our cnostructs didn't work. Or we can test our repressors, promoters and inducers and have a systematic approach to anaylizing our system so that when something works or doesn't work we will know why. Thus we have designed a few tests which should give us relative and absolute values of our system that we can then plug into the model.

We decided to to test for three values. The combined transcription/translation rate of each repressor, the cooperativity of each repressor and the concentration of ligand needed to deactivate each repressor. We managed to design three tests all using the same constructs so as to minimize ligations. These tests should determine our variables independantly, i.e. changing synthesis rate should not change cooperativity of repression.


Synthesis Rate

The combined transcription/translation rate of the repressor is the combined strength of the promoter and the RBS. In
The Alpha test Architecture
our model this is the alpha value. Our model predicts that the alpha values for each repressor should be fairly comparable (the stable region is along the 1 to 1 to 1 line in 3D). Since we can't change the promoter strength very easily, we will change the RBS strength to obtain similar alpha values for all repressors.







Cooperativity of Repression

The cooperativity describes an inherent characteristic of a repressor's repression. In our system we want to know how
The Beta test Architecture
much increased repressor concentration will increase repression. In our model, cooperativity is an exponent, beta, so that the repressor concentration is raised to the beta ([repressor]^beta = total repression). For beta = 1, repression increases linearly with repressor concentration. With twice as much repressor there is twice the repression. For beta = 2, repression increases with the square of the concetration. Twce as much repressor leads to four times the repression. Our model predicts that our system will be more robust with greater alpha values and the tri stable region (in the graph) will be larger. Beta must be larger than one for the system to be stable.




Ligand Concentration

Naturally, we don't want to add more ligand than we need. In what ever application the project might find, if we wanted to change the state again by adding a different
The Ligand test Architecture
ligand, we wouldn't want excessive amounts of the first ligand floating around.


Retrieved from "http://2007.igem.org/wiki/index.php/Tristable/Testing_Constructs"