- We need a standardized units to compare expression in different chassis.
- Ideal unit for this is PoPS (Polymerase per Second), however this cannot directly be measured. Currently the best approach is to measure indirectly using Rate of GFP molecules sysnthesised sec-1 CFU-1 (CFU meaning Colony forming units which is a measure of cells)
- We would like to use this approach, however, there is no CFU in in-vitro and in-veso. We need to change CFU so we can relate it to in-vitro and in-veso. Several approaches:
- Units in terms of molecules involved -Units relate to key enzymes i.e. Ribosome and RNA poymerase and relate to key reactants i.e.amino acids, RNA molecules, DNA. However, requires lots of units because for a given set of conditions do not know the key rate determining molecule. In terms of our project we feel this is too ambitious.
- Units not in terms of cells, but rather in terms of a cells 'expression system'. We then define a cell as a volume that has a defined level and ratio of expression machinery.
- Error involved - Literature tells us the volume of a cell is ~10-15 L and so the potential error in our experiment is massive.
- Need to normalise several key parameters that make our unit less generic
- DNA levels per aqueous cell volume.
- Non cellular concentrations and ratios will be found in vitro, how to compare this to a cell to allow for comparison.e.g.the potential gene expression per aqueous cell volume is changing.
- Complexity of what we are attempting.
Two key issues how generic is this, can anyone use out unit or is it too limiting and does this allow the chassis to be compared.
List what we plan to characterize that are chassis dependent characteristics e.g. temp, life span of chassis
Construct Dependent Characteristics
List what we plan to characterize that are construct dependent characteristics e.g. change of expression.