IGEM:IMPERIAL/2007/Notebook/2007-8-24

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7

In Vitro Testing of pLux 25oC
Tested for the working condition of the DNA construct pTet-LuxR-pLux-GFP. This experiment will carry on until fluorescence reaches that of the control. Protocol can be found here under Phase 1-In vitro testing on the experimental design page. Results can here under Results on the experimental design page

Preparation of Reaction Buffer for S30 Cell Extract

 * Phosphoenolpyruvate has been added
 * 30 &micro;l of buffer aliquotes put into eppendorf tubes
 * Home-made cell extract (S30) ready to be tested
 * Reaction mixture:
 * S30 cell extract 16.2 &micro;l
 * Reaction buffer 30 &micro;l
 * Puruvate kinase 3.1 &micro;l
 * rNTPs 1 &micro;l
 * DNA 4 &micro;l
 * ddH2O 5.7 &micro;l
 * Total volume: 60 &micro;l

In Vitro Testing of pTet 25oC using Home-Made Cell Extract with Commercial Pre-Incubation Mix
Tested for the viability of the home-made cell extract. Protocol followed was according to the Promega one, and can be found here under Phase 1-In vitro testing on the experimental design page. Results: It was observed that no fluorescence was produced during the incubation period of over 4 hours.

In Vitro Testing of pTet 25oC using Home-Made Cell Extract and Reaction Buffer
Tested for the viability of the home-made cell extract and reaction buffer. Protocol followed was according to the one in the paper. Results: It was observed that no fluorescence was produced during the incubation period of over 4 hours.

Vesicles Formation with GFP
Formation of Vesicles

Using POPC/dodecane suspension from the day before, two samples were prepared:
 * 2ml of suspension was taken to prepare an interface according to protocol.
 * 250&mu;l of 100x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
 * Special care was taken to protect the GFP solution from light at all times.

In addition, another suspension was prepared using Span-80 and mineral oil. The Span-80 was simply added to the mineral oil and mixed for 10 minutes with a magnetic stir bar before 250&mu;l of 100x diluted GFP solution was added to it.

3 samples prepared:
 * Sample 1: 2ml of POPC/dodecane/GFP emulsion, with no previously prepared interface.
 * Sample 2: 1ml of POPC/dodecane/GFP emulsion added over interface prepared with 2ml of POPC/dodecane suspension.
 * Sample 3: 2ml of Span-80/mineral oil/GFP emulsion, with no previously prepared interface.

All samples were centrifuged at 120x g.

Samples from both the POPC/dodecane and Span-80/mineral oil emulsions were also collected for observation under the microscope.

Results
 * Sample 1: Vesicles observed, but very sparse, with no fluorescence.
 * Sample 2: Numerous small vesicles encapsulating GFP were observed.
 * Sample 3: Vesicles observed, but with no fluorescence. Vesicles were very mobile.
 * POPC/dodecane Emulsion: Fluorescent esicles observed, but very sparse.
 * Span-80/mineral oil Emulsion: Numerous small vesicles encapsulating GFP were observed.

Preparations

No preparations were carried out.