IGEM:IMPERIAL/2007/Notebook/2007-8-16

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/15 view=threemonths format=%name/%year-%month-%day weekstart=7

Preparation of Cell Extract

 * 1) O.D. of overnight culture was 3.417 at 9am (still less than mid-log phase of 0.D.600 = 4.5)
 * 2) Decided to harvest cells anyway
 * 3) Prepared s30 cell extract
 * 4) *Added protease inhibitors to the cell extract
 * 5) *Might add RNAse inhibitors when preparing the next cell extract if this one does not work

Protocols can be found at S30/S12 Cell Extract in the general protocols page

Testing of DNA Constructs

 * 1) Cells containing constructs pTet-GFP and pT7-GFP and pTet-LuxR-pLux-GFP were incubated overnight at 37°C
 * 2) Tested for viability of construct pTet-GFP in vivo. Conclusion: Works
 * 3) Tested for viability of construct pTet-LuxR-pLux-GFP in vivo. Conclusion: Works
 * 4) Tested for viability of construct pT7-GFP in vivo. Conclusion: Uncertain.
 * 5) *We realised that the E.coli cells were not yet induced to produce T7 promoter.
 * 6) *200µl IPTG was added to 600µl of the cells, and placed in a 37°C incubator overnight
 * 7) *Testing for this construct will be done tommorrow
 * pcI-GFP was not tested in vivo because construct was not ready.

Pilot Preparation of Vesicles

 * Formation of Vesicles: The suspension prepared the day before was used to form vesicles.
 * 1) 2ml of suspension was used to prepare an interface (according to the protocol)
 * 2) 200x diluted GFP solution was used to prepare the emulsion
 * 3) Two samples were prepared:
 * 4) *One following the protocol, with 2ml suspension interface and 100&mu;l of emulsion added
 * 5) *One using 2ml of emulsion to form the interface, without further addition of material


 * Results: Very few vesicles were observed under a light microscope with phase contrast. The fluorescence microscope did not produce any results.


 * Preparations: The desiccation and suspension stages of the vesicle preparation protocol were carried out once again.
 * 1) Two 100ml beakers were prepared with 125&mu;l of DOPC in 50ml of mineral oil
 * 2) The suspensions were sonicated for 30mins each
 * 3) *At this point, it was found that the desiccator had no vacuum, and a further two suspensions were prepared as described above
 * 4) A total of four beakers, two well-desiccated and two poorly-desiccated, were produced
 * 5) These were left overnight in a 27&deg;C incubator