Imperial/Wet Lab/Lab Notebook/2007-08-24
From 2007.igem.org
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= 24 August 2007 = | = 24 August 2007 = | ||
+ | |||
+ | ==In Vitro Testing of pLux 25<sup>o</sup>C== | ||
+ | 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. | ||
+ | <br><br> | ||
+ | Protocol can be found here under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.2 Phase 1-In vitro testing] on the experimental design page. | ||
+ | <br><br> | ||
+ | Results can here under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Results_2.2 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 | ||
+ | *#ddH<sub>2</sub>O 5.7 µl | ||
+ | *Total volume: 60 µl | ||
+ | |||
+ | |||
+ | ==In Vitro Testing of pTet 25<sup>o</sup>C using Home-Made Cell Extract with Commercial Pre-Incubation Mix== | ||
+ | Tested for the viability of the home-made cell extract. | ||
+ | <br><br> | ||
+ | Protocol followed was according to the Promega one, and can be found here under [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.1 Phase 1-In vitro testing] on the experimental design page. | ||
+ | <br><br> | ||
+ | Results: It was observed that no fluorescence was produced during the incubation period of over 4 hours. | ||
+ | |||
+ | ==In Vitro Testing of pTet 25<sup>o</sup>C using Home-Made Cell Extract and Reaction Buffer== | ||
+ | Tested for the viability of the home-made cell extract and reaction buffer. | ||
+ | <br><br> | ||
+ | Protocol followed was according to the one in the paper. | ||
+ | <br><br> | ||
+ | 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 [[Imperial/Wet Lab/Lab Notebook/2007-08-23|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. | ||
+ | |||
+ | <br> | ||
+ | {|align="left" border="1" | ||
+ | | width="200px"| [[image:IC07_image116.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image114.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image114b.jpg|200px]] | ||
+ | |- | ||
+ | |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. | ||
+ | |- | ||
+ | | width="200px"| [[image:IC07_image120.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image118.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image118b.jpg|200px]] | ||
+ | |- | ||
+ | |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. | ||
+ | |}<br clear="all"> | ||
+ | |||
+ | {|align="left" border="1" | ||
+ | | width="200px"| [[image:IC07_image102.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image103.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image104.jpg|200px]] | ||
+ | |- | ||
+ | |colspan="3" width="600px"|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. | ||
+ | |}<br clear="all"> | ||
+ | |||
+ | {|align="left" border="1" | ||
+ | | width="200px"| [[image:IC07_image110.jpg|200px]] | ||
+ | | width="200px"| [[image:IC07_image109.jpg|200px]] | ||
+ | |- | ||
+ | |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"> | ||
+ | |||
+ | |||
+ | '''Preparations''' | ||
+ | |||
+ | No preparations were carried out. | ||
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Latest revision as of 21:05, 26 October 2007
24 August 2007
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.
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.