Part:BBa_J37027

Cre/lox Pops Blocker

Motivation

We wanted a way of controlling the activation of the positive-feedback loop in our [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser predator-prey based oscillator]. The idea was to use some kind of switch. Because the available switches in the registry had certain drawbacks, we decided to design our own switch. Consequently, we came up with the Cre-Lox Pops Blocker system.

Part Pre Activation

Part Post Activation

Note : DNA is removed

Overview

This part is designed to be placed downstream of a promoter and prevent any Pops from the Promoter passing through this part. It will do this until an accompanying Cre Recombinase plasmid becomes activated. Once the Cre recombinase is activated the enzyme produced will permanently cut a section of DNA from the plasmid containing this part. This short section of DNA contains stop codons. Hence, once these are removed the polymerase can pass through this part and transcribe downstream genes. This short section of DNA is then degraded.

This is useful if a component of the system must be grown up but not activated until a certain external stimulus is added, for example, a positive feedback loop.

This part should be very efficient at preventing Pops passing through it.

The part also contains a RFP reporter which is transcribed in the 3’-5’ direction. This means that un-activated parts will fluoresce red and activated parts will not fluoresce. This allows you to see that the part is working in your system. It also allows you to observe the efficiency of activation of the part in your system.

Because of the way Cre recombinase works, the excised reporter will remain in a small plasmid and continue to be transcribed for a short time. This plasmid, however, will not have an origin of replication. Hence, it will not be copied and the fluorescent protein should stop being produced after around 15min.

Many Thanks to Jonny Wells and David Mann for helpful suggestions