24 August 2007

In Vitro Testing of pLux 25^oC

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 µl of buffer aliquotes put into eppendorf tubes
• Home-made cell extract (S30) ready to be tested
• Reaction mixture:
  1. S30 cell extract 16.2 µl
  2. Reaction buffer 30 µl
  3. Puruvate kinase 3.1 µl
  4. rNTPs 1 µl
  5. DNA 4 µl
  6. ddH₂O 5.7 µl
• Total volume: 60 µl

In Vitro Testing of pTet 25^oC 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 25^oC 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.
• 250μl 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 250μl 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.

Microscope pictures of vesicles in Sample 2, formed from 1ml POPC-dodecane-GFP emulsion. Left: A vesicle under white light. Centre: The same vesicle, with fluorescence. Right: The same picture as the one in the centre, but enhanced with gamma correction and full saturation.
More vesicles formed from Sample 2. Left: A group of vesicles under white light. Centre: The same vesicles, with fluorescence. Right: The same picture as the one in the centre, but enhanced with gamma correction and full saturation.

Vesicles made using Span-80 and mineral oil. Left: A group of vesicles in the Span80-Mineral Oil-GFP emulsion (not in aqueous solution). Centre and Right: Another group of vesicles in Sample 3, formed from the Span80-Mineral Oil-GFP emulsion on the left.

Microscope pictures of a small vesicle in Sample 2, formed from 1ml POPC-dodecane-GFP emulsion. The vesicle was actually moving very quickly. Left: The vesicle under white light. Right: The same vesicle, with fluorescence (open the larger image to see the fluorescence more clearly).

Preparations

No preparations were carried out.