Team:Cambridge/Notebook/Week5
From 2009.igem.org
Categories :
Project :
-
Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
Week 5
Monday
Dry Work
Violacein Synthesis
Used GeneDesigner from DNA2.0 to plan what we wanted synthesised, taking into account the different genes in the operon. This would be synthesised with codon optimisation for E. coli. All forbidden restriction sites have been removed (EcoRI, XbaI, SpeI, PstI, NotI)
Wet Work
=Protocol de-bugging
For the amplification project, we need to amplify pSB3K3 as a low-copy number plasmid vector in order to transfer the activator constructs into low copy number plasmid. We tried 4 different PCR reaction mixtures, and used a Zymogen PCR wash kit before running them on an 0.8% Agarose E-gel. After looking at the gel, the ladder was quite smeared and no PCR product was visible. It was found that the samples run on the gel had a very low DNA concentration. However, some PCR solution was left before the wash kit was used, and using the Nano-Drop it was determined that it had a high concentration of DNA, so the PCR worked, we just need to refine the protocols after PCR.
Amplification
Mini-prepped I746374, I746375, I746380, and I746391; incubated overnight cultures of the rest of the 11 activator constructs in preparation for miniprep.