IGEM:IMPERIAL/2007/Calendar/2007-9-7
From OpenWetWare
We are in Week 9 of the iGEM project
Project Calendar
<calendar> name=IGEM:IMPERIAL/2007/Calendar date = 2007/08/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>
Meeting with Prof Freemont
Experiments
Initial testing:
constructs tested in-vivo and in-vitro
- pTet - find out why doesn't work below 10°C and above 45°C
- pT7 - need to reclone (doesn't have the right RBS)
- pLux - doesn't work below 1nM
Tests for working conditions:
- ID:
- Test on construct 1 carried out with various [AHL] - works for 3,5,7,10,50nM. Reaches steady state within about 3 and a 1/2 hours
- Carrying out purification of LuxR for construct 2
- Need to do a titration curve for different [AHL]
- CBD: Problems with results - steady state not reached within 8 hours (lab time). Tried staggering but miss the steady state - data not useful
- Try for longer access to lab and fluorometer
- Get a 24hr fluorometer
Also problem with data - fluorescence increases after overnight incubation
- dsRed express - vector should be cloned by next week
- Solution to GFP problem:
- Prioritise experiments with dsRed express
- Try to find purified GFP mut-3b
- dsRed2 to be cloned - in case other options don't work out
- Other problems with data:
- Mixing the samples - find out whether better to shake or not. Can't centrifuge as affects reaction
- Evaporation - oil to prevent it, but might stop O2 supply too. Or can get a rate of evaporation for different temperatures and account for it using modelling
Vesicles
- Put in cell extract - fluorescence seen but may not be GFP, can be background fluorescence
- Need to do a control experiment - as not sure if expression actually heppening inside the vesicles (contact a Chem. engineer)
- A lot of fluorescence seen outside the vesicles
- Can be due to vesicles popping or GFP not actually getting into the vesicles
- Emulsion process - doesn't seem good enough to encapsulate the cell extract
- Characterise the process of emulsion - will take 3-4 days
- Vesicles seem quite stable - last for ~ 3 days
- Permeability - without it expression worse than in-vitro and signal really weak
- No permeability improves duriblity of DNA -
- May only get non-permeable vesicles
- Will get measure of population activity rather than individual as not possible to characterise the size of the vesicles, even with the same protocols
Modelling
- Deterministic models complete
- Currently doing data analysis
- Programming a curve fitting program to extract parameters
- 24hr experiments required to fully analyse data
- Need kinetic constants from data - currently a lot of assumptions
Meeting with other teams
- Vincent to sort out a meeting with Cambridge during term time
Other
- Registry - must only submit the parts actually being used in the project
- Have no name yet!