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Team:Paris Liliane Bettencourt/Notebook/2011/08/12/
From 2011.igem.org
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.
Cyrille
Minipreps and purification of pHM3
The transformation was observed in the morning. The ones from gel extraction (GE) where very few, whereas the ones from the minipreps where very numerous. We took two clones from the GE, and 1 from the MP, and growth them overday for a miniprep and two glycerols.
The miniprep was done and measured by tecan. Due to the problems of purity we observed before, we loaded the samples on a gel, alone and digested by both EcoRI and EcoRV. Only the more pure plasmid will be keppee, and the others tubes throw away. The miniprep where not that pure, because some of them had bad ratio.
The runned gel gives:
Preparation of clean pHM3 without any modification
From the transformation done the dahy before yesterday, 3 minipreps and the 3 corresponding glycerols where done.
The result of the miniprep is loaded on a gel, both normal and digested by EcoRI. This is to check the purity of the sample before exchanging the glycerol that has been done before.
The gel gives:
This is not cleaner than what we expected, eventhough, two of the transformation come from a very diluted transformation. If we look at the other gel of the experience above, we meet the same problems: the weels 6 contains the plasmid from the gel extraction of the lower band and eventhough, there are upper bandes.
They can be attributed to a protein that binds the dna or supper coiling differences.
Kevin
TECAN DL Digested PCR Purificated products
- 1.4 : 8,1 ng - 1.5 : 7,5 ng
- 2.4 : 8,8 ng - 2.5 : 6,2 ng
- 3.7 : 5,9 ng - 3,8 : 5,4 ng
- 4,7 : 6,5 ng - 4,8 : 6 ng
Ligation pSB1C3 - DL PCR Digested products
- 1uL 10X T4 Buffer
- 0,5uL T4 Ligase
- 1,6uL Vector pSB1C3 XS Digested (20ng)
- 2,5uL Insert 1 and 3 or 3uL Insert 2 or 4
- qsp H20 10uL
Let 1h at 22,5°C.
Transformation of ligation products
Usual protocol
