IGEM:IMPERIAL/2007/Projects/Cell by date/TestingValidation

Cell by Date: Testing/Validation

• Introduction
• Specifications
• Design
• Modelling & Implementation
• Testing/Validation
• Notes
• References

Experimental design

• Temperature increases the rate of reactions - increased temperature means faster gene expression over a given time.
• System only deals with protein synthesis and its reporter function - a visual signal.
• Coupling firefly genes with that of a well-characterized consitutive promoter, response is good.
• We will then calibrate the synthesis of fluorescent protein (which is easier as opposed to calibrating cell density) with that of the predicted shelf-life (date label).
  • Assuming the cold chain process only occurs at 4oC, it will lead to the predicted outcome.
  • Any temperature increases, and over a threshold would lead to over the range fluorescence.
  • Degradation of protein is minimal with fine-tuning of cell free medium characterization
  • Proof of principle of the application of CFE.

Fluorescent Signal

• The first problem is that a fluorescent signal will only be visible if a cell population is at a certain density. This is because there needs to be a great enough density of cells expressing a fluorescent protein so that there is a strong enough signal to be visible by eye. This will be a problem if the cells are exposed to varying conditions early on in the sell by date when cell population is low.
• The fluorescent proteins expressed will also need to be relatively stable from degradation. This is because if we initiate the expression of the fluorescent proteins then we want the signal to be sustained so that our 'cell-by-date' has a memory.
  • A further point on this is that we can't use GFP as a reporter in hypoxic conditions GFPHypoxic
  • Possible solution to this problem is to use a reporter with a long Half-Life so that there is enough time for someone to see the signal being outputted by our system. EGFP Seems to be one such reporter having a half life that may exceed 24hours1. Enocding vector found for EGFP that places it under control of Plac promoter4. DsRed seems to be another possible reporter to use3.

• In order to avoid complication in design we have opted to just use one reporter, RFP, instead of using two eg. transition from GFP to RFP when problems occour. This is due to time constraints - it make take time for the RFP to overcome the GFP.