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Team:BCCS-Bristol/Notebook/Week 8
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week 8
- pQE31-GFP was digested and ligations with digested and purified FhuA were performed.
- XL1-Blue competent cells were transformed with the pQE31-GFP+FhuA ligations.
- The concentration of the DNA parts that we are going to send for sequencing was determined by spectrophotometry. These parts include: AraC-RBS, AraC-RBS-FhuA/OsmE, GFP-terminator and bioscaffolds-GFP-terminator.
- Liquid cultures of the bioscaffolds-GFP-terminator were prepared and minipreps performed to isolated the plasmid DNA. Then the whole bioscaffold-GFP-terminator constructs were purified by gel extraction and ligations with the AraC-RBS-FhuA/OsmE were prepared.
- Overnight ligations set-up;
1) AraC-RBS-FhuA-Bioscaffold-GFP-Terminator 2) AraC-RBS-OsmE-Bioscaffold-GFP-Terminator
- Prepare liquid cultures for pQE31-GFP-FhuA.
Promoter-RBS-GFP-terminator ligation
- This will be used to check that GFP works and also to check if the AraC promoter works by trying to control its level of activity using arabinose.
- Restriction digest and gel purify GFP-terminator part and then ligate it to the already cut AraC-RBS on pSB1A2 plasmid.
The figure illustrates the construct AraC-RBS-GFP-TErminator created to assess promoter activity. The ladder is 1kb DNA marker from NEB. Lanes run in triplets with Double Digest (XbaI/Osme), Single Digest(XbaI) and Uncut versions of DNA plasmids carrying the construct. Presence is illustrated by the bands created in the double digest lane (top:plasmid backbone bottom:construct)
