Team:BCCS-Bristol/Notebook/Week 10

From 2009.igem.org

Revision as of 13:26, 9 October 2009 by Pm5527 (Talk | contribs)

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.




The ladder is standard 1kb NEB DNA marker. Lanes run in quadruplets illustrating Uncut, Single Digest with BpuEI, Single Digest with XbaI and Double digest with XbaI and BpuEI of plasmid DNA carrying AraC-RBS-FhuA-Bioscaffold-GFP-Term on a pSB2K3 plasmid. Lanes 1-4 show pre-mutated DNA of the construct. It is apparent that lanes with BpuEI cause star activity for prolonged periods of incubation, exceeding 60min. Successful ligations and mutations are shown by lanes 5-8 where only two bands are produced in double digests.


: Lanes run in quadruplets illustrating Uncut, Single Digest with BpuEI, Single Digest with XbaI and Double digest with XbaI and BpuEI of plasmid DNA carrying AraC-RBS-FhuA-Bioscaffold-GFP-Term on a pSB2K3 plasmid. Lanes 1-4, 25-27 show successful ligations and also succesful removal of the BpuEI site in GFP that conflicted the Biosca�ffold specifi�c enzyme.
Retrieved from "http://2009.igem.org/Team:BCCS-Bristol/Notebook/Week_10"