Team:BCCS-Bristol/Notebook/Week 10

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(New page: {{:Team:BCCS-Bristol/Header}} {{:Team:BCCS-Bristol/NotebookHeader}} ===Week 10=== ====Bioscaffold Tests==== * The LacZ reporter gene seems not to be cut by XbaI enzyme and hence canno...)
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No GFP was seen to be produced in both the control and the FhuA-GFP construct. Also the FhuA-GFP construct seem not to be of high concentration.
No GFP was seen to be produced in both the control and the FhuA-GFP construct. Also the FhuA-GFP construct seem not to be of high concentration.
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[[Image:BCCS_cell_conc.jpg|center|500px|frame|The graph shows on the y-axis Optical Density at absorbance of 600nm (representative of cell concentration). The higher the value, the higher the cell concentration. The x-axis is the percentage of L-Arabinose used for promoter induction. ARF is AraC-RBS-FhuA-GFP and results show drop in optical density after promoter induction. ARG is AraC-RBS-GFP-Terminator and after promoter induction there is no drop in optical density. Clearly the promoter (AraC-RBS) works whilst the FhuA-GFP protein fusion inhibits cell growth.]]

Revision as of 13:10, 9 October 2009

BCCS-Bristol
iGEM 2009


Week Zero Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12


Week 10

Bioscaffold Tests

  • The LacZ reporter gene seems not to be cut by XbaI enzyme and hence cannot be used in the Alternative Bioscaffold tests.
  • To replace LacZ we decided to use RFP (E1010) as a downstream protein for the Bioscaffold alternative tests. Ligations and transformations were set up for the 4 different bioscaffold versions, upstream of RFP on the pSB2k3 plasmid backbone. Only A1(CspCI st.) and A2(BseRI) were succesful.
  • Site-Directed mutagenesis was set up to mutate the illegal EcoRI site in OsmE and the Bioscaffold illegal site in GFP. This was performed only on the full constructs shown below (* indicates mutagenesis site): 
1) AraC-RBS-FhuA-Bioscaffold-GFP*-Terminator (for 4 different bioscaffold versions)
2) AraC-RBS-OsmE*-Bioscaffold-GFP-Terminator (for 4 different bioscaffold versions)
  • Transformations with XL-1 Blue were set up for the above mutants. Transformations with 2ul of the mutated products were unsuccessful.
  • Transformed versions A1(CspCI St.) A2(BseRI) A3(CspCI alternative) of the bioscaffold constructs with FhuA. 7ul in Nova-Blue strain. Transformations were successful but only few colonies appeared.

Alternative Assembly Constructs

  • A finished version of alternative assembly was created featuring: 
1) AraC-RBS-FhuA-GFP
  • The finished construct was tested by setting up overnight 5ml liquid cultures in LB Broth (Ampicillin only). The following was set up: 
1) AraC-RBS-FhuA-GFP
2) AraC-RBS-GFP-Term (control and promoter testing)

In the morning 100ul aliquots were transferred in fresh medium (5ml LB+Ampicillin) and were induced with arabinose in varying concentrations; 

1) 0%
2) 0.05%
3) 0.1%
4) 0.2%
5) 0.5%
6) 1%

No GFP was seen to be produced in both the control and the FhuA-GFP construct. Also the FhuA-GFP construct seem not to be of high concentration.


The graph shows on the y-axis Optical Density at absorbance of 600nm (representative of cell concentration). The higher the value, the higher the cell concentration. The x-axis is the percentage of L-Arabinose used for promoter induction. ARF is AraC-RBS-FhuA-GFP and results show drop in optical density after promoter induction. ARG is AraC-RBS-GFP-Terminator and after promoter induction there is no drop in optical density. Clearly the promoter (AraC-RBS) works whilst the FhuA-GFP protein fusion inhibits cell growth.
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