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

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We are in Week 9 of the iGEM project

Project Calendar

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30


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!
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