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Team:Paris Liliane Bettencourt/Notebook/2011/08/12/
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===GFP monitor=== | ===GFP monitor=== | ||
| + | Because of troubles with the Phyperspank and the progress in the cloning of the T7 amplification system, a monitor construct will be made containing: pT7 + spoVG + GFP + Ter T7. It will be used to verify the functionality of the tRNA amber suppressor. | ||
Revision as of 16:31, 12 August 2011
Contents |
Danyel & Camille
tRNA
Due to uncertainties as to the eventual funcionality of the tRNA amber suppressor, we have decided to make several constructs:
1A) promotor Pveg + RBS (spoVG) + tRNA + Ter (B0015) 1B) Pveg + RBS + tRNA
P hyperspank + RBS + tRNA + Ter P hyperspank + RBS + tRNA
However, we have abandoned the constructs with the hyperspank promotor, because we received the wrong part from the registry.
Though ideally it would be best to not have an RBS preceding the gene, all promotor bricks already have an RBS attached to it. We have also verified that there is no ATG or TTG (the most used start codons in B.subtilis : "Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis" Eduardo P. C. Rocha, Antoine Danchin and Alain Viari). There is a GTG codon (the least used start codon) at the 14 nucleotide of the gene sequence.
The part we received in place of the hyperspank promotor is a Pveg + RBS (different from spoVG). We decided to build two constructs with the same logic as the 1A) and 1B). They are temporarily named 4A) and 4B)
All parts were appropriately digested and are ready for ligation.
T7 amber
Sequencing results showed successful results for the T7 amber quickmutation. The T7 has been appropriately digested along with the Terminator B0015. The two will be ligated and transformed in the following days.
GFP monitor
Because of troubles with the Phyperspank and the progress in the cloning of the T7 amplification system, a monitor construct will be made containing: pT7 + spoVG + GFP + Ter T7. It will be used to verify the functionality of the tRNA amber suppressor.
