"
Team:BCCS-Bristol/Notebook/Week 9
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Contents |
Week 9
- Sequencing results indicate that our fusions were corrent and we are working with the correct proteins/DNA parts.
Constructing a simple fusion to test the promoter
- XL1-Blue compet<math>Insert formula here</math>ent cells transformed with plasmid containing AraC-RBS-GFP-Terminator insert.
Alternative Assembly method (a.k.a. Quick and Dirty)
- Minipreps of cultures of cells containing pQE31 with FhuA-GFP insert, restriction digests and gel electrophoresis were performed to certify that the insert of interest is there.
- FhuA-GFP insert was then cut and isolated using gel extraction and ligations were prepared with open pSB1A2 containing the AraC-RBS.
- Tranformations of XL1-Blue with pSB1A2 containing AraC-RBS-FhuA-GFP (ligations) were done. Moreover liquid cultures of cells containing the AraC-RBS-GFP-termninator insert were prepared.
- DNA from the AraC-RBS-FhuA-GFP liquid cultures was miniprepped and assayed with restriction enzymes to test for the presence of the construct.
Bioscaffold Assemblies
* AraC-RBS-FhuA-Bioscaffold-GFP-Terminator * AraC-RBS-OsmE-Bioscaffold-GFP-Terminator
- The constructs above were used to transform XL1-Blue cells.
- Transformations of the Bioscaffold assemblies above were successful. Restrikes and liquid cultures were set up.
- Restrikes and liquid cultures were successful. DNA was miniprepped from the liquid cultures and assayed with restriction enzymes to test for the presence of the entire construct. Results showed that the construct was successfully incorporated in the plasmids.
- Site directed mutagenesis needs to be done to mutate an EcoRI site in OsmE and a BpuEI site in GFP. The former in order to make OsmE RFC10 compatible and the latter in order to be able to use the Bioscaffold.
- Primers for site-directed mutagenesis were designed and ordered.
Alternative Test for the Bioscaffold
- To be able to characterize the action of the Bioscaffold a simple construct of two proteins would be created with the Bioscaffold in the middle and the relevant assembly taking place.
- For this purpose we are going to try and use again LacZ reporter gene to be the protein downstream of the Bioscaffold. If the process works then the colonies would appear blue in X-Gal plated agar plates.
