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| - | {{:Team:BCCS-Bristol/Header}}
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| - | {{:Team:BCCS-Bristol/NotebookHeader}}
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| - | ===Week 10===
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| - | ====Bioscaffold Tests====
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| - | * The LacZ reporter gene seems not to be cut by XbaI enzyme and hence cannot be used in the Alternative Bioscaffold tests.
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| - | * 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.
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| - | * 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):
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| - | 1) AraC-RBS-FhuA-Bioscaffold-GFP*-Terminator (for 4 different bioscaffold versions)
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| - | 2) AraC-RBS-OsmE*-Bioscaffold-GFP-Terminator (for 4 different bioscaffold versions)
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| - | * Transformations with XL-1 Blue were set up for the above mutants. Transformations with 2ul of the mutated products were unsuccessful.
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| - | * 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.
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| - | ====Alternative Assembly Constructs====
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| - | * A finished version of alternative assembly was created featuring:
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| - | 1) AraC-RBS-FhuA-GFP
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| - | * The finished construct was tested by setting up overnight 5ml liquid cultures in LB Broth (Ampicillin only). The following was set up:
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| - | 1) AraC-RBS-FhuA-GFP
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| - | 2) AraC-RBS-GFP-Term (control and promoter testing)
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| - | In the morning 100ul aliquots were transferred in fresh medium (5ml LB+Ampicillin) and were induced with arabinose in varying concentrations;
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| - | 1) 0%
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| - | 2) 0.05%
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| - | 3) 0.1%
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| - | 4) 0.2%
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| - | 5) 0.5%
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| - | 6) 1%
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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.
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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.]]
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