Team:Edinburgh/Notebook/Green light producer


These notes correspond to the part of the project detailed here.

Green Light Producer


PCR of the Edinburgh iGEM 2009 part to remove the pYeaR nitrite responsive promoter.
Blunt ended ligation. Retransform into JM109 and grow on amplicillin.


Sequence for the brighter luciferase was sent to Geneart for synthesis.

PCR was done to remove the extra 6 bases between the RBS and the coding sequence. Forward primer started with the start codon. Reverse primer started with the fusion scar then the RBS. Blunt ended ligation and transformation followed.


LUCIE luciferase sequence has just arrived from geneart. Primers to PCR the part out of the geneart vector were ordered.


PCR was run to remove part. Product was purified, digested, and ligations into pSB1C3 set up.


White colonies were selected from the plates and subcultured.


Liquid cultures were set up for miniprep.


Minipreps of the colonies were done, digested and run on a gel. Clones 2 and 3 look good.


Preps were digested with EcoRI and XbaI. J33202 was digested with EcoRI and SpeI. After purificationm ligations were set up.


Transformation of yesterday's ligations.


Wiki freeze is today, so liquid cultures were prepared in the morning without subculturing to check for glowing. Cells do glow well, but are not as green as hoped. The mutations seem to introduce a red shift.