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Team:BCCS-Bristol/Notebook/Week 7
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week 7
- Transform XL1-Blue with the 4 ligations (pQE31+GFP, AraC-RBS + FhuA, AraC-RBS + OsmE, GFP-terminator).
- Transformations with AraC-RBS + FhuA/OsmE worked but NOT for pQE31-GFP and GFP-terminator.Repeat transformations for the ones that didn't worked.
- Trasformation for GFP-Terminator worked and liquid cultures were prepared.
- pQE31+GFP trtansformation didn't worked. Fresh stock of pQE31 was cut open, phosphatase treated and purified. PCR set up to amplify new stocks of GFP to be ligated to the new stock of pQE31. Ligations between pQE31 and GFP with varying ratios of plasmid vector to GFP insert were carried out.
i.e. Plasmid: Insert
1:1
1:2
1:3
1:4
1:5
- pSB1A2 plasmids containing AraC-RBS-FhuA and AraC-RBS-OsmE were extracted using minipreps and later were cut with restriction enzymes and phosphatase treated. The first part of the conventional method (with bioscaffold) is finally completed.
- Minipreps were performed for the GFP-terminator-containing cultures and the plasmid on which they are found was digested with restriction enzymes to allow the incorporation of the bioscaffold, which is the next step.
- XL1-Blues were transformed with the pQE31+GFP ligations with the varying ratios of vector to insert. Moreover the bioscaffold arrived and its DNA was used to transform XL1-Blues.
- Both of the above transformations worked, liquid cultures were set up and used to carry out minipreps.
- The 4 different bioscaffold versions were cut with restriction enzymes and isolated using the gel extraction protocol. At the end of the day ligations between the cut GFP-terminator and the 4 bioscaffold versions were set up.
Ladder is 1kb DNA marker. Lanes run in triplets running in Double Digest (XbaI/OsmE), Single Digest(XbaI) and Uncut versions of the DNA plasmid carrying the AraC-RBS-FhuA construct.
Ladder is 1kb DNA marker. Lanes run in triplets running in Double Digest (XbaI/OsmE), Single Digest(XbaI) and Uncut versions of the DNA plasmid carrying the AraC-RBS-OsmE construct.
Ladder is 1kb DNA marker. Lanes run in triplets running in Double Digest (XbaI/OsmE), Single Digest(XbaI) and Uncut versions of the DNA plasmid carrying the GFP terminator construct.
Ladder is 1kb DNA marker. Lanes run in triplets running in Double Digest (XbaI/OsmE), Single Digest(XbaI) and Uncut versions of the DNA plasmid carrying the different Bioscaffold Versions.
