Team:NTNU Trondheim/Experiments and Results
From 2012.igem.org
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==Experiments and results== | ==Experiments and results== | ||
- | To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page]. | + | To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page]. |
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Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; | Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; | ||
- | + | * a protein coding brick for colicin E1, | |
- | + | * a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, | |
- | + | * the lld promotor + RBS from ''E.coli'', | |
- | + | * the lld promotor + RBS from ''C.glutamicum''. | |
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work. | This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work. | ||
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===Colicin (<partinfo>BBa_K822002</partinfo>)=== | ===Colicin (<partinfo>BBa_K822002</partinfo>)=== | ||
- | Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. | + | Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used: |
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+ | {|border="1" | ||
+ | !Primer | ||
+ | !Sequence | ||
+ | |- | ||
+ | |Colicin fwd | ||
+ | |GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta | ||
+ | |- | ||
+ | |Colicin rev | ||
+ | |GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa | ||
+ | |} | ||
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+ | To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. | ||
Revision as of 22:01, 26 September 2012