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Team:Valencia/prototypes
From 2012.igem.org
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- Artifical terminator with %T≈85<br> | - Artifical terminator with %T≈85<br> | ||
- 6bp stem, 4nt loop<br> | - 6bp stem, 4nt loop<br> | ||
| + | |||
| + | <strong>Constructing the BioBrick</strong> | ||
| + | We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). | ||
| + | We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here. | ||
</p> | </p> | ||
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Revision as of 22:14, 26 September 2012
Designed Biobricks
Designed BioBrick
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the psbAI promoter which binds to the lambda promoter inhibiting the luxI transcription, when psbAI is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.
Parts Information:
BBa_K754000 (link): S. elongatus PCC7942 psbAI promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.
BBa_Q04510 (link): Consists in the next parts:
- B0034: RBS
- C0051: cI repressor from E. coli phage lambda
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.
- B0015: double terminator
This is the most commonly used terminator. It seems to be reliable
-R0051: promoter (lambda cI regulated)
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.
BBa_C0061 (link): Autoinducer synthetase for AHL
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
BBa_B1002 (link): Terminator
- Artifical terminator with %T≈85
- 6bp stem, 4nt loop
Constructing the BioBrick
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo).
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.
