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Team:BCCS-Bristol/Notebook/Week 4
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Week 4
- One more try to get Fiu protein on pSB1A3 backbone. NovaBlue transformed with ligation products of pSB1A3 and Fiu.
- Grew colonies for FhuA and OsmE midi preps.
- Carried out midi preps for FhuA and OsmE ( [FhuA]=378.9 ng/ul, [OsmE]=113.2 ng/ul).
- Transformed Nova Blue with biobrick part BBa_B0014 (double terminator)
- Culture colonies of BBa_B0014 transformants to get ready for mini prep.
- PCR FhuA and GFP for assembling together a quick and dirty fusion to be ready prior to BioScaffold arrival.
- Carried out colony growth (agar & liquid) for the AraC promoter (2 parts BBa_R0080 (lacking O2 region) and BBa_R0081 (the O2 region).
- Miniprepped DNA from cultures of BBa_R0080 and R0081.
- Attempted a 2-way ligation and of the BBa_R0080 and R0081 parts. The final product should be a 2-component biobrick on a plasmid in 5'-R0081-R0080-3' direction which can be transformed into cells.
- R0081 part was purified by gel extraction after being treated by double digest. R0080 and R0081 were ligated together and used to transform NovaBlue cells.
- First attempt to make the AraC promoter from the two parts failed.
- Fiu still not being ligated on ANY backbone.
Standard 1kb NEB Ladder. Lanes 1-2 FhuA Midipreps, Lanes3-4 OsmE midipreps.
Standard 1kb NEB Ladder. Lanes 1-2 FhuA PCR fragment for Quick and Dirty assembly Method, Lanes3-4 GFP PCR fragments for quick and dirty assembly method.
The figure indicates the presence of the AraC promoter subunits. Lanes 1-6 show the unsuccesful ligation of the Fiu Carrier protein. Lanes 7-9 illustrate Double Digest with XbaI and SpeI,Single Digest with XbaI and Uncut versions of the plasmid carrying R0080. Similarly lanes 10-12 show the same for R0081. The promoter fragments are 149bp and 183bp respectively and do not appear on the gel but the presence is illustrated by the band shift between the Double and Single Digests.
