"
Team:BCCS-Bristol/Notebook/Week 3
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week3
- Ligation successful only for the OsmE and FhuA inserts on pSb1A3 backbone. Unfortunately we couldn't get any of the 3 proteins on the pSB1A2 backbone.
- Seems that overxpression of these proteins on pSB1A2 is unfavourable for the cells. Hence decided to replace the existing promoter with an inducible one of our choice such as the AraC promoter.
- Promoter removed from pSB1A2 and religated. Fiu still not able to be ligated on any of the two backbones.
- In the meantime the two parts comprising of the AraC promoter were extracted from the iGEM kit plates and used to transform NovaBlue competent cells. Also liquid cultures of the transformed cells were prepared.
- Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.
- Finding canditate primers for sequencing the first 3 carrier biobrick proteins (fhuA/osmE/fiu).
- Started thinking of a quick and dirty in-frame fusion for testing functional carrier-reporter gene fusions.
- Found easy fusion way for in line testing. Will take out end of FhuA and scar formed after RFC10 assembly of FhuA-GFP using RE's to take out FhuA end ( including TAA TAA SCAR) and part of GFP start. Will replace lost coding sequences with PCR.
- Ordered primers for the quick-n-dirty assembly method.
