IGEM:IMPERIAL/2006/project/popsblocker/Design

Pops Blocker

• Specifications
• Design
• Modelling
• Implementation
• Validation

Design

Two devices are required:

• 1) A complementary sequence for a reporter with loxP sites either side
• 2) Cre recombinase under the control of a promoter

The device J37027 can be found here

Design of primers and method of construction

Proposed Implementation of Cre-LoxP system

Cre recombinase

Cre recombinase device in registry J37030

Sequence and primers here

There is one further step that we need to take if we want to successfully incorporate this system into the whole system. The plasmid containing the Cre enzyme coding region will be going into the prey cell. However the prey cell will already contain the main coding plasmid, which has ampicillin resistance. Therefore to ensure that the second plasmid is taken up into the cell as well, we need to swap plasmids by ligation. Ligating into a kanamycin resistant plasmid is probably our best bet. We could do this after the device has been constructed in the ampicillin resistant plasmid.

LoxP sites

The two LoxP sites that we are going to use are Lox66 and Lox71 - mutant LoxP sites

Sequences - Lox66 and Lox71

Alterations to Cre-Lox System

Further Reading

• Cre recombinase: The universal reagent for genome tailoring
• Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein