IGEM:IMPERIAL/2006/project/popsblocker: Difference between revisions

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'''[[IGEM:IMPERIAL/Methodology/Part_documentation_template | Use this template]]'''
===Cre-LoxP System===


==Cre-LoxP System==
==Motivations==


*We wanted a way of controlling the activation of the positive-feedback loop in our predator-prey based oscillator. The idea was to use some kind of switch, and seeing as the available switches in the registry all had certain drawbacks, we went about designing our own and what we came up with was the Cre-LoxP system
*The use of Cre recombinase to excise regions of DNA between two loxP sites could be incorporated into part [http://parts.mit.edu/registry/index.php?title=Part:BBa_J37015 J37015]. Our aim is to insert two loxP sites into this part, which, when in place, would block the synthesis of any genes downstream of it, and when removed would allow their synthesis. Thus the Cre-loxP system would effectively act as a switch/Pops blocker controlled by the activity of the promoter in front of the Cre enzyme.
 
*In theory, using Cre recombinase as a switch/Pops blocker appears to be a very efficient and solid method.


The use of Cre recombinase to excise regions of DNA between two loxP sites could be incorporated into part [http://parts.mit.edu/registry/index.php?title=Part:BBa_J37015 J37015]. Our aim is to insert two loxP sites into this part, which, when in place, would block the synthesis of any genes downstream of it, and when removed would allow their synthesis. Thus the Cre-loxP system would effectively act as a switch controlled by the activity of the promoter in front of the Cre enzyme. 
==Part interface==


*Using Cre recombinase as a switch appears to be, in theory, a very efficient and solid method.  
 
*The parts are not available in the registry however, which means that we would have to make them.  
{| border="0" width="100% align="center"
*The loxP sites are around 20 bases in length and so sequences can be be worked out and bought fairly easily.  
|-
*The Cre recombinase site is around a kilobase in length. However we have been able to obtain the enzyme from one of the labs, and as it is isolated in a plasmid, this will make it much simpler for us to incorporate it into our design.
|
*The Cre recombinase will also need a promoter - perhaps we could use one such as lacI? (we need to find out whether IPTG has any effect on other promoters though)
{| border="1" cellpadding="15" width="100%"
*With all the parts available, we estimate that the length of time for implementation should be under a week.
|-
! style="background:#66CC99"| INPUTS !!style="background:#FF3333"| OUTPUTS
|-
| Cre ||rowspan = "2"| Reporter
|-
| Pops
|}
||
[[Image:IGEM_IMPERIAL_Methodology_Specifications_Cre.PNG|right|350px]]
|}
 
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Latest revision as of 09:47, 30 October 2006


Cre-LoxP System

Motivations

  • We wanted a way of controlling the activation of the positive-feedback loop in our predator-prey based oscillator. The idea was to use some kind of switch, and seeing as the available switches in the registry all had certain drawbacks, we went about designing our own and what we came up with was the Cre-LoxP system
  • The use of Cre recombinase to excise regions of DNA between two loxP sites could be incorporated into part J37015. Our aim is to insert two loxP sites into this part, which, when in place, would block the synthesis of any genes downstream of it, and when removed would allow their synthesis. Thus the Cre-loxP system would effectively act as a switch/Pops blocker controlled by the activity of the promoter in front of the Cre enzyme.
  • In theory, using Cre recombinase as a switch/Pops blocker appears to be a very efficient and solid method.

Part interface

INPUTS OUTPUTS
Cre Reporter
Pops

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