IGEM:IMPERIAL/2007/Notebook/2007-8-24: Difference between revisions
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'''Results''' | '''Results''' | ||
* Sample 1: | * Sample 1: Vesicles observed, but very sparse, with no fluorescence. | ||
* Sample 2: | * Sample 2: Numerous small vesicles encapsulating GFP were observed. | ||
* Sample 3: | * Sample 3: Vesicles observed, but with no fluorescence. Vesicles were very mobile. | ||
* POPC/dodecane Emulsion: | * POPC/dodecane Emulsion: Vesicles observed, but very sparse, with no fluorescence. | ||
* Span-80/mineral oil Emulsion: | * Span-80/mineral oil Emulsion: Numerous small vesicles encapsulating GFP were observed. | ||
<br> | |||
{|align="left" border="1" | {|align="left" border="1" | ||
| width="200px"| [[image:IC07_image116.jpg|200px]] | | width="200px"| [[image:IC07_image116.jpg|200px]] | ||
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| width="200px"| [[image:IC07_image114b.jpg|200px]] | | width="200px"| [[image:IC07_image114b.jpg|200px]] | ||
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|colspan="3" width="600px"|Microscope pictures of vesicles 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. | |colspan="3" width="600px"|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. | ||
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| width="200px"| [[image:IC07_image120.jpg|200px]] | | width="200px"| [[image:IC07_image120.jpg|200px]] | ||
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|colspan="3" width="600px"|More vesicles formed from | |colspan="3" width="600px"|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. | ||
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| width="200px"| [[image:IC07_image104.jpg|200px]] | | width="200px"| [[image:IC07_image104.jpg|200px]] | ||
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|colspan="3" width="600px"|Vesicles 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 aqueous buffer, formed from the Span80-Mineral Oil-GFP emulsion on the left. | |colspan="3" width="600px"|Vesicles found in Sample 3, 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 aqueous buffer, formed from the Span80-Mineral Oil-GFP emulsion on the left. | ||
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| width="200px"| [[image:IC07_image109.jpg|200px]] | | width="200px"| [[image:IC07_image109.jpg|200px]] | ||
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|colspan="2" width="400px"|Microscope pictures of a small vesicle 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). | |colspan="2" width="400px"|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). | ||
|}<br clear="all"> | |}<br clear="all"> | ||
Revision as of 07:24, 5 September 2007
<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>
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 µl of buffer aliquotes put into eppendorf tubes
- Home-made cell extract (S30) ready to be tested
- Reaction mixture:
- S30 cell extract 16.2 µl
- Reaction buffer 30 µl
- Puruvate kinase 3.1 µl
- rNTPs 1 µl
- DNA 4 µl
- ddH2O 5.7 µl
- Total volume: 60 µl
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.
- 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.
In addition, samples from both the POPC/dodecane and Span-80/mineral oil emulsions were 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: Vesicles observed, but very sparse, with no fluorescence.
- Span-80/mineral oil Emulsion: Numerous small vesicles encapsulating GFP were observed.
Preparations
No preparations were carried out.