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Team:BCCS-Bristol/Notebook/Week 5
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week 5
- The double terminator BBa_B0014 was taken out of the iGEM kit plates and used to transform NovaBlue cells.
- Minipreps and restriction digests were performed for terminator cultures (B0014) to check that they have the correct insert.
- Ligations set up between R0080 (on pSB1A2) and R0081 and used to transform NovaBlue competent cells.
Alternative Assembly Method
- In this method we will use standard non-iGEM-procedures to fuse FhuA and GFP. This construct will be placed downstream of an AraC-RBS fusion and upstream of a terminator. The final assembly product will be RFC10 compliant.
- The aim is to check whether a cargo protein like GFP will be transported to vesicles by one of our carrier proteins of interest like FhuA. It should be completed more quickly than the other method(at least in principle).
- It can also be used as a back up in case the bioscaffold method does not work.
- In general a PCR-amplified GFP fragment will be inserted in a pQE31 plasmid. Then a PCR-amplified FhuA fragment will be inserted upstream of the GFP.
- The whole construct will be cut and ligated upstream of an already made AraC-RBS fusion and at the end a terminator will be inserted upstream.
- Performed restriction digests of pQE31 and GFP PCR product.
- Make liquid cultures of NovaBlue transformed with complete AraC promoter.
- PCR reactions to amplify FhuA, GFP and LacZ. GFP, FhuA and pQE31 are cut with appropriate enzymes for subsequent reactions and then purified.
- LacZ reactions didn't work. We will proceed only with GFP.
- Minipreps for colonies with Arac promoter.
- Ligations set up between:
1. AraC and RBS 2. pQE31 and GFP
1kb NEB Standard Ladder. Lanes run in triplets featuring Double Digest (XbaI/SpeI), Single Digest(XbaI) and uncut versions of DNA plasmid carrying the AraC promoter. The promoter is seen as a single band at the bottom of the Double Digest lanes. The top band is the pSB1A2 Carrier plasmid.
1kb NEB Standard Ladder. Lanes run in triplets featuring uncut versions of DNA plasmid carrying the Terminator B0014, Single Digest(XbaI) and Double Digest (XbaI/SpeI). The terminator is seen as a single band at the bottom of the Double Digest lanes. The top band is the pSB1A2 Carrier plasmid.
