2019 Vilnius-Lithuania iGEM team chose to charaterize Part:BBa_K861173, mRFP generator controlled by PcstA (a glucose-repressible promoter). In this construct, PcstA is activated by CRP (cAMP receptor protein) and cAMP complex, therefore, RFP is expressed at low glucose concentration.
Our measurement differs from others done for PcstA (for example, with Part:BBa_K200018 and Part:BBa_K729008 ), since we measured growth kinetics with a microplate reader. Points were taken every 30 minutes for 14 hours and were able then to plot mRFP fluorescence divided by OD 600 against time. Also, characterization focused on different glucose concentrations (5%, 2%, 1% 0.4%, 0.1%, 0.01%, 0.001% and 0%) instead of varying carbon sources, the carbon source was changed to glycerol (0.4%) for one measurement.
We found a clear correlation between the glucose concentration in the media and the maximum amount of mRFP expression.A 9 fold difference was observed between bacteria, grown in M9-CA, containing 5% and 0% glucose. Also, a difference in output, when grown in glucose and glycerol containing media can be seen, which can be explained by the difference in by the metabolic pathways used by the tested cells.
The graph showing change in mRFP expression in time with different glucose concentrations (5%, 2%, 1% 0.4%, 0.1%, 0.01%, 0.001%) and glycerol (0.4%). A 9 fold difference can be seen between bacetria grown in 5% and 0% glucose media
Measuring with a microplate reader is one of the most useful methods for characterizing how synthetic pathways work. For the measurements, the iGEM community protocol suggests using LB media. From our experience, a significant drawback is its vast autofluorescence, which restricts measurement precision, especially for low expression systems. Additionally, performing measurements in M9 media allows more control in varying the growth environment.
What is more, we show that measuring systems dynamics has superior precision compared to single-point measurements and lead to easier application of data to a mathematical model. In the case of this part characterization, knowing the change in output by varying the glucose concentration allows the part to be used in controlling the protein of interest level in the cell.
Measuring kinetics may also seem as more troublesome, since vastly more data is collected, however, writing R code that can be easily adapted different experiments and measurements reduces the difficulty in processing and visualizing the data.