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

We are in Week 9  of the iGEM project

Project Calendar
name=IGEM:IMPERIAL/2007/Calendar date = 2007/08/01 view=threemonths format=%name/%year-%month-%day weekstart=7

Initial testing:
constructs tested in-vivo and in-vitro
 * pTet - find out why doesn't work below 10&deg;C and above 45&deg;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!