Team:Paris Liliane Bettencourt/Notebook/2011/08/12/

From 2011.igem.org

(Difference between revisions)
(tRNA)
(tRNA)
Line 15: Line 15:
They are temporarily named 4A) and 4B)
They are temporarily named 4A) and 4B)
-
All parts were appropriately digested and are ready for ligation.
+
All parts have been appropriately digested and are ready for ligation.
===T7 amber===
===T7 amber===

Revision as of 16:37, 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 according to "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 according to the same article) 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 have been 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. The pT7 and the spoVG + GFP + Ter T7 construct have been appropriately digested and are ready for ligation.