Part Improvement
We improved the part BBa_0430, an EYFP output device, by joining it with a constitutively expressed (by ptet, BBa_R0040) ECFP gene, to be used as a control for normalization of the output measurement, as this control gene would be subject to the same influences as the reporter gene (eg. cell density, plasmid copy number, metabolic load, environment properties). This method was used following the efforts of Rudge et al. on characterizing intrinsic promoter properties.
This fluorescent protein pair is good for this type of measurement due to their spectral separation. In our experiments, we used excitation/emission values of 430/480 for CFP and 500/530 for YFP. The CFP protein has an LVA degradation tag, which should make the CFP measure more accurate as a representation of the immediate state of the gene's activity, independing of non-degraded, previous values.
Unfortunately, this makes the CFP overall fluorescence lower and creates the need for a more precise measurement, rather than just measuring from LB. We have obtained good results while using LB and centrifuging the samples then ressuspending them in H2O, and while growing the cells in M9 medium.
Our new part is BBa_2771020, which still preserves de modularity property of the original part, as other teams can insert promoter parts upstream of our part and readily have a good reporter gene for the activity of one promoter.