IGEM:IMPERIAL/2007/Calendar/2007-9-7

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