Team:Groningen/project memory units

From 2011.igem.org

(Difference between revisions)
(Memory Unit)
 
(24 intermediate revisions not shown)
Line 1: Line 1:
{{HeaderGroningen2011}}  
{{HeaderGroningen2011}}  
-
=== Memory Unit ===
+
=Memory Units=
 +
As memory units, we are using bistable autoinducing loops that can be suppressed via a reversed antisense promoter (jammer). The autoinducing loop will act as an amplifier of the input signal with the promoter inducing expression of its own transcription factor. To switch off the loop, we adopted the endogenous device from the 2009 iGEM team of British Columbia, which implemented a reverse antisense promoter downstream of the coding sequence (https://2009.igem.org/Team:British_Columbia/Jammer).
-
<br>Our system composed of 2 memory units, consisting autoinducing loop and jammer-based off switch. Autoinducing loop play a role as an amplifier of input signal by regulating its own expression. To switch it off, we adopted the endogenous device from British Columbia 2009 team, which uses a reverse antisense promoter downstream a coding sequence (https://2009.igem.org/Team:British_Columbia/Jammer).  
+
[[File:bistable.png|thumb|500px|center|Mechanism of bistability in autoinducing loops.]]
-
<br>First memory unit contain cI gene downstream modified lambda PRM promoter (Bba_I12006) and flanked-terminators in forward and reverse direction. This system is induced by CI (lambda repressor) and repressed by LasR. Whereas, second memory unit contain lasR gene downstream LasB promoter (BBa_R0079), which induced by LasR and repressed by arabinose.  
+
==Our implementation==
 +
The first memory unit is based on a modified lambda Prm promoter (BBa_I12006) producing its own inducer cI. By means of the reversed antisense lasB promoter it will be repressed in the presence of LasR. The second memory unit consists of the lasB promoter (BBa_R0079) producing its own induction factor LasR. Both autoinducing loops can be repressed by an arabinose inducible reverse antisense pBAD promoter. The transcription factor genes (cI and LasR) were fused in various combinations with different RBS and degradation tags, resulting in 12 different construct variants. These enable us to study the dynamics of expression and half-life of different autoinducing loop variants in order to subsequently determine the constructs that best meet the criteria for bistability.
-
<br>To study the dynamic expression and half-life of autoinducing loop components, which best fitted the expected system, the transcription factor gene (cI and LasR) was fused in combination with various RBS and degradation tag, resulting 12 different constract variants.  
+
{|
 +
|[[File:unit1.png|thumb|300px|Lambda cI based autoinducing loop.]]
 +
|[[File:unit2.png|thumb|300px|LasR based autoinducing loop.]]
 +
|}
-
<br>Firstly, we would like to test the leakage of promoter by cotransform autoinducing loop plasmid and reporter plasmid under control of appropriate promoter. Secondly, cotransformation assembled input and output systems with plasmid bearing various-variants of autoinducing loops would provide information about memory unit behaviour in time. Finally, integration of experimental data and our model would lead us to fine-tuned the expected system behaviour.
+
==Experimental Planning==
 +
 
 +
First, we want to test promoter leakage by cotransforming a plasmid containing the autoinducing loop and a reporter plasmid under the control of an appropriate promoter. Second, we will cotransform assembled input and output systems with plasmids bearing various variants of autoinducing loops. These constructs will provide us with information about memory unit behavior in time. Finally, integration of experimental data in our model will allow us to fine-tune the expected system behavior.
 +
 
 +
 
 +
 
 +
{{FooterGroningen2011}}

Latest revision as of 00:01, 22 September 2011


Memory Units

As memory units, we are using bistable autoinducing loops that can be suppressed via a reversed antisense promoter (jammer). The autoinducing loop will act as an amplifier of the input signal with the promoter inducing expression of its own transcription factor. To switch off the loop, we adopted the endogenous device from the 2009 iGEM team of British Columbia, which implemented a reverse antisense promoter downstream of the coding sequence (https://2009.igem.org/Team:British_Columbia/Jammer).

Mechanism of bistability in autoinducing loops.

Our implementation

The first memory unit is based on a modified lambda Prm promoter (BBa_I12006) producing its own inducer cI. By means of the reversed antisense lasB promoter it will be repressed in the presence of LasR. The second memory unit consists of the lasB promoter (BBa_R0079) producing its own induction factor LasR. Both autoinducing loops can be repressed by an arabinose inducible reverse antisense pBAD promoter. The transcription factor genes (cI and LasR) were fused in various combinations with different RBS and degradation tags, resulting in 12 different construct variants. These enable us to study the dynamics of expression and half-life of different autoinducing loop variants in order to subsequently determine the constructs that best meet the criteria for bistability.

Lambda cI based autoinducing loop.
LasR based autoinducing loop.

Experimental Planning

First, we want to test promoter leakage by cotransforming a plasmid containing the autoinducing loop and a reporter plasmid under the control of an appropriate promoter. Second, we will cotransform assembled input and output systems with plasmids bearing various variants of autoinducing loops. These constructs will provide us with information about memory unit behavior in time. Finally, integration of experimental data in our model will allow us to fine-tune the expected system behavior.