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Team:BCCS-Bristol/Notebook/Week 11
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week 11
Alternative Assembly Constructs
- Last week's experiment was repeated with ARabinose induction occuring overnight. Again the FhuA-GFP construct seemed not to be growing.
- Cell concentration assays revealed that the fusion construct was inhibiting E.coli growth. The only GFP control seemed to be growing normally. (see results here)
- To test the inhibition fresh transformants of :
1) AraC-RBS-OsmE 2) AraC-RBS-FhuA 3) AraC-RBS-FhuA-GFP 4) AraC-RBS-GFP-Terminator
were set up. 3 colonies of each were picked and grown overnight. The next day 20ml cultures were set-up for each colony and growth was tested with A600 O.D. every half an hour (pre and post arabinose induction).
- Results indicate that construct 1 and 3 lyse the cells.
Bioscaffold Tests =
- To test for successful mutation 3 colonies of each mutated construct (BpuEI site mutated out) were miniprepped and tested with BpuEI. Unfortunately the enzyme was not working.
- To test the Bioscaffold the mutated construct of AraC-RBS-FhuA-Bioscaffold-GFP was transferred to the pSB2k3 backbone that is compatible with the Bioscaffold specific enzymes. (enzymes used by the bioscaffold A2(BseRI and BpuEI) do not appear in this backbone.
- As a back-up plan, if the mutation was unsuccessful the C-terminal region of GFP will be cut out in order to assess Bioscaffold efficiency.
The graph illustrates the assessment of growth post promoter induction with L-Arabinose at 0.1%. The y-axis is time(min) and the x-axis log10 A600 Optical Density, representing cell concentration. Promoter induction occurred at 95min. ARG is AraC-RBS-GFP-Terminator, ARFG is AraC-RBS-FhuA-GFP ARO is AraC-RBS-OsmE and ARF is AraC-RBS-FhuA. ARO and ARFG indicate drop in OD A600 whilst ARF and ARG keep growing normaly post-induction.
