Team:Warsaw/results

From 2009.igem.org

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{{WarHead1|lab=none|project=none|team=none|modelling=none}}
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<b><h1>lacI/cI bistable switch</h1></b>
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==lacI/cI bistable switch==
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 +
Control of cell invasion and further operation of our system - the BacInVader is done by two bistable switches. The first one controls expression of invasion genes.
 +
 
 +
===Design===
 +
 
 +
The structure of this switch is shown below:
 +
 
 +
<partinfo>BBa_K177038 DeepComponents</partinfo>
 +
 
 +
The most important components of the switch are repressor proteins cI (denoted here as cIts, see explanation below) and lacI. The whole device is composed
 +
of two main parts:
 +
 
 +
LacI under control of 'cI lam' promoter
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<partinfo>BBa_K177012 DeepComponents</partinfo>
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and
 +
 
 +
cI under control of Plac promoter
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<partinfo>BBa_K177011 DeepComponents</partinfo>
 +
 
 +
===How it works?===
 +
 
 +
Because cI is repressor of 'cI lam' promoter and lacI is repressor of Plac promoter (denoted here as 'LacI') there are only two mutually exclusive states of the switch:
 +
 
 +
*Plac promoter is active which leads to cI expression. CI binds to 'cI lam' promoter and represses it so no genes under this promoter are expressed.
 +
*'cI lam' promoter is active which leads to LacI expression. LacI binds to Plac promoter and represses it.
 +
 
 +
According to this, state of the switch should be stable in time until some external stimulus is applied.
 +
 
 +
===How to switch its state?===
 +
 
 +
To set the state of the switch 0,1mM IPTG and high temperature (42<sup>o</sup>C) are used.
 +
 
 +
*After addition of 0,1mM IPTG LacI gets inactivated and transcription from Plac starts. cI is expressed which leads to repression of 'cI lam' promoter.
 +
*Heating the bacteria in 42<sup>o</sup>C inactivates CI protein and starts expression from 'cI lam' promoter.
 +
 
 +
===Experimental results===
 +
 
 +
To test if our assumptions are correct we have assembled switch testing device <partinfo>BBa_K177038</partinfo>. In this device we have connected GFP with Plac dependent part of the switch and mRFP with 'cI lam' dependent part. After transformation of the complete construct at 30<sup>o</sup>C random distribution of green and red colonies was observed. It was expected because we haven't used temperature nor IPTG to set the initial state of the switch.
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 +
==termosensitive version of lambda cI protein==
 +
 
 +
==selection of strains compatible with switch==
-
In order to function properly, the switch requires a termosensitive version of cI protein. As it turned out, the cI encoding brick from the registry did not contain the termosensitive mutation (verified by sequencing). The switch constructed with the wild-type cI was unfunctional, expressing only RFP, with slight variation in intensity, depending on the temperature. A point mutation has been introtuced into the completed device, in order to ensure termosensitivity of cI. As a result, a population of bacteria with new properties has been obtained. The switch expresses GFP in 30 and 37 <sup>o</sup>C, regardless of the presence of IPTG in the medium. In 42 <sup>o</sup>C on the other hand, the reporter expressed is exclusively RFP. Having been activated by high temperature, the expression of RFP continues in 30 <sup>o</sup>C for some time, gradually switching back to GFP. The rate of this process can be increased by an addition of IPTG.
 
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{{WarFoot1}}

Revision as of 15:38, 21 October 2009

Contents

lacI/cI bistable switch

Control of cell invasion and further operation of our system - the BacInVader is done by two bistable switches. The first one controls expression of invasion genes.

Design

The structure of this switch is shown below:


The most important components of the switch are repressor proteins cI (denoted here as cIts, see explanation below) and lacI. The whole device is composed of two main parts:

LacI under control of 'cI lam' promoter


and

cI under control of Plac promoter


How it works?

Because cI is repressor of 'cI lam' promoter and lacI is repressor of Plac promoter (denoted here as 'LacI') there are only two mutually exclusive states of the switch:

  • Plac promoter is active which leads to cI expression. CI binds to 'cI lam' promoter and represses it so no genes under this promoter are expressed.
  • 'cI lam' promoter is active which leads to LacI expression. LacI binds to Plac promoter and represses it.

According to this, state of the switch should be stable in time until some external stimulus is applied.

How to switch its state?

To set the state of the switch 0,1mM IPTG and high temperature (42oC) are used.

  • After addition of 0,1mM IPTG LacI gets inactivated and transcription from Plac starts. cI is expressed which leads to repression of 'cI lam' promoter.
  • Heating the bacteria in 42oC inactivates CI protein and starts expression from 'cI lam' promoter.

Experimental results

To test if our assumptions are correct we have assembled switch testing device . In this device we have connected GFP with Plac dependent part of the switch and mRFP with 'cI lam' dependent part. After transformation of the complete construct at 30oC random distribution of green and red colonies was observed. It was expected because we haven't used temperature nor IPTG to set the initial state of the switch.

termosensitive version of lambda cI protein

selection of strains compatible with switch

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