"
Team:RMIT Australia/Project
From 2010.igem.org
(→Creation of the Taq Polymerase Part) |
m (→Making the Inducible T7 Expression Vector) |
||
| Line 429: | Line 429: | ||
=== Making the Inducible T7 Expression Vector === | === Making the Inducible T7 Expression Vector === | ||
| - | The aim for this section is to create | + | The aim for this section is to create an inducible expression backbone. This will be achieved by altering the backbone biobrick '''BBa_J04500''', by removing the -35 and -10 elements (promoter) and inserting the bacteriophage T7 promoter. The strategy used to will be Quikchange XL mutagenesis to alter the promoter elements into restriction sites: |
* -35 --> AflII | * -35 --> AflII | ||
* -10 --> BamH1 | * -10 --> BamH1 | ||
| Line 435: | Line 435: | ||
These restriction sites are NOT found in the BBa_J04500 part or vector (pSB1A2), so there will be no cross cutting. | These restriction sites are NOT found in the BBa_J04500 part or vector (pSB1A2), so there will be no cross cutting. | ||
| - | + | The T7 promoter is small enough for it to be constructed from primers. Two ligation ends would be designed to match the sticky ends of the restriction sites introduced. The T7 can thus be inserted as the promoter of the system, being regulated by IPTG (LacO site) and glucose/cAMP (CAP-binding site). | |
== Results == | == Results == | ||
Revision as of 02:50, 7 July 2010
Overall project
Your abstract
Project Details
Creation of the Taq Polymerase Part
Making the Inducible T7 Expression Vector
The aim for this section is to create an inducible expression backbone. This will be achieved by altering the backbone biobrick BBa_J04500, by removing the -35 and -10 elements (promoter) and inserting the bacteriophage T7 promoter. The strategy used to will be Quikchange XL mutagenesis to alter the promoter elements into restriction sites:
- -35 --> AflII
- -10 --> BamH1
These restriction sites are NOT found in the BBa_J04500 part or vector (pSB1A2), so there will be no cross cutting.
The T7 promoter is small enough for it to be constructed from primers. Two ligation ends would be designed to match the sticky ends of the restriction sites introduced. The T7 can thus be inserted as the promoter of the system, being regulated by IPTG (LacO site) and glucose/cAMP (CAP-binding site).
