Imperial/Wet Lab/Lab Notebook/2007-08-28
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+ | ==In vitro Testing of pTet-LuxR-pLux-GFP at room temperature== | ||
+ | Testing of the construct was done at a low concentration of AHL 0.1nM to emulate a real biofilm. This experiment tests whether the system is sensitive enough to respond to the low amounts of AHL. | ||
+ | <br><br> | ||
+ | Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.2|Phase 1-In vitro testing]] on the experimental design page. | ||
+ | <br><br> | ||
+ | Results: Very low levels of fluorescence was observed. | ||
+ | |||
+ | ==Construction of pT7-GFP== | ||
+ | |||
+ | #Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight | ||
+ | #Spun down cells and minipreped | ||
+ | |||
+ | Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Miniprep|Miniprep]] in the general protocols page | ||
+ | |||
+ | *[Vector DNA] = 137.9 ng/μl | ||
+ | *[Insert DNA] = 987.1 ng/μl | ||
+ | |||
+ | #Set up digestion to cut out insert and vector | ||
+ | #*DNA (5 μl insert/ 15 μl vector) | ||
+ | #*Nde 0.75 μl | ||
+ | #*BamH1 0.75 μl | ||
+ | #*10X BSA 2 μl | ||
+ | #*10X BamH1 Buffer 2 μl | ||
+ | #*ddH20 to make up total volume of 20 μl | ||
+ | #Digestion left at 37°C for 1 hour | ||
+ | |||
+ | ==Vesicles== | ||
+ | '''Preparations''' | ||
+ | |||
+ | Two suspensions were prepared for the next day: | ||
+ | # 125μl of POPC in 50ml of dodecane | ||
+ | # 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil | ||
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Revision as of 14:04, 10 October 2007
Contents |
28 August 2007
In vitro Testing of pTet-LuxR-pLux-GFP at room temperature
Testing of the construct was done at a low concentration of AHL 0.1nM to emulate a real biofilm. This experiment tests whether the system is sensitive enough to respond to the low amounts of AHL.
Protocol can be found here under Phase 1-In vitro testing on the experimental design page.
Results: Very low levels of fluorescence was observed.
Construction of pT7-GFP
- Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight
- Spun down cells and minipreped
Protocols can be found at Miniprep in the general protocols page
- [Vector DNA] = 137.9 ng/μl
- [Insert DNA] = 987.1 ng/μl
- Set up digestion to cut out insert and vector
- DNA (5 μl insert/ 15 μl vector)
- Nde 0.75 μl
- BamH1 0.75 μl
- 10X BSA 2 μl
- 10X BamH1 Buffer 2 μl
- ddH20 to make up total volume of 20 μl
- Digestion left at 37°C for 1 hour
Vesicles
Preparations
Two suspensions were prepared for the next day:
- 125μl of POPC in 50ml of dodecane
- 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil