IGEM:IMPERIAL/2006/project/popsblocker/Design: Difference between revisions
No edit summary |
No edit summary |
||
Line 15: | Line 15: | ||
==Registry== | ==Registry== | ||
*insert part logo | *insert part logo | ||
Cre recombinase device in registry [http://parts.mit.edu/registry/index.php/Part:BBa_J37030 J37030] | |||
==Design choices== | ==Design choices== | ||
==Design== | ==Design== | ||
Line 77: | Line 52: | ||
*[http://www3.interscience.wiley.com/cgi-bin/fulltext/70001850/PDFSTART Cre recombinase: The universal reagent for genome tailoring] | *[http://www3.interscience.wiley.com/cgi-bin/fulltext/70001850/PDFSTART Cre recombinase: The universal reagent for genome tailoring] | ||
*[http://nar.oxfordjournals.org/cgi/content/full/30/17/e90 Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein] | *[http://nar.oxfordjournals.org/cgi/content/full/30/17/e90 Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein] | ||
{| border="1" width="100%" | |||
|- style="background:lightgreen" | |||
! INPUTS !! biological component !! comments | |||
|- | |||
| input 1 || bla bla bla || bla bla bla | |||
|- | |||
| input 2 || bla bla bla || bla bla bla | |||
|- | |||
| input 3 || bla bla bla || bla bla bla | |||
|- style="background:orange" | |||
! OUTPUTS !! biological component !! comments | |||
|- | |||
| output 1 || bla bla bla || bla bla bla | |||
|- | |||
| output 2 || bla bla bla || bla bla bla | |||
|} | |||
==Open issues== | |||
*The parts are not available in the registry however, which means that we would have to make them. | |||
*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) | |||
*With all the parts available, we estimate that the length of time for implementation should be under a week. |
Revision as of 10:43, 22 October 2006
Cre-LoxP System
Registry
- insert part logo
Cre recombinase device in registry J37030
Design choices
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
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
INPUTS | biological component | comments |
---|---|---|
input 1 | bla bla bla | bla bla bla |
input 2 | bla bla bla | bla bla bla |
input 3 | bla bla bla | bla bla bla |
OUTPUTS | biological component | comments |
output 1 | bla bla bla | bla bla bla |
output 2 | bla bla bla | bla bla bla |
Open issues
- The parts are not available in the registry however, which means that we would have to make them.
- 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)
- With all the parts available, we estimate that the length of time for implementation should be under a week.