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Team:UC Chile/Cyanolux/Future
From 2012.igem.org
Juan Alamos (Talk | contribs) |
Juan Alamos (Talk | contribs) |
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<h1>Promoters Redesigned</h1> | <h1>Promoters Redesigned</h1> | ||
| - | We are designing new versions of our chosen promoters. Now we have changed the promoter construction methodology in terms of the lenght of the upstream sequence considered. | + | We [https://2012.igem.org/Team:UC_Chile/Results/LuxBrick#N-decanal_Effect_Characterization_-_.28BBa_K325909_and_BBa_K325905.29 have seen] that even if induced with substrate, our luciferase constructs don´t show luminescence, though the constructs´ sequences are verified and well characterized luciferases do show light emition. |
| + | Our advisors in cyanobacteria have told us that sometimes promoter sequences can be placed several hundred of bases upstream the +1 site, even inside protein coding sequences. | ||
| + | That´s why we are designing new versions of our chosen promoters. Now we have changed the promoter construction methodology in terms of the lenght of the upstream sequence considered. | ||
Instead of considering just 150-200bp, up to 1000bp will be amplified from Synechocystis genome. | Instead of considering just 150-200bp, up to 1000bp will be amplified from Synechocystis genome. | ||
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<h1>Transcriptional verification</h1> | <h1>Transcriptional verification</h1> | ||
| - | Even if our promoters are o.k, colony PCRs can´t tell us wether the transcriptional machinery of our cyanobacteria is recognizing our constructs | + | Even if our promoters are o.k, colony PCRs can´t tell us wether the transcriptional machinery of our cyanobacteria is recognizing our constructs. |
We are designig new primers to make RT-PCRs that unmistakably verify transcription of the Lux genes at the specified hours. | We are designig new primers to make RT-PCRs that unmistakably verify transcription of the Lux genes at the specified hours. | ||
Revision as of 03:37, 27 September 2012
Contents |
Promoters Redesigned
We have seen that even if induced with substrate, our luciferase constructs don´t show luminescence, though the constructs´ sequences are verified and well characterized luciferases do show light emition. Our advisors in cyanobacteria have told us that sometimes promoter sequences can be placed several hundred of bases upstream the +1 site, even inside protein coding sequences. That´s why we are designing new versions of our chosen promoters. Now we have changed the promoter construction methodology in terms of the lenght of the upstream sequence considered. Instead of considering just 150-200bp, up to 1000bp will be amplified from Synechocystis genome.
Transcriptional verification
Even if our promoters are o.k, colony PCRs can´t tell us wether the transcriptional machinery of our cyanobacteria is recognizing our constructs. We are designig new primers to make RT-PCRs that unmistakably verify transcription of the Lux genes at the specified hours.
Cyrcadian expression characterization
We have allready built part [http://partsregistry.org/Part:BBa_K743018 BBa_K743018] to characterize differential expression of sfGFP at different moments of the day. Other promoters (new ones or those allready built) will be placed by means of Gibson assembly controlling the expression of this reporter.
New Biosafety strategies
We have allready assembled part [http://partsregistry.org/Part:BBa_K743010 psb1C3_IntS] wich has a double goal: to insert LuxCDEG genes into Synechocystis and to confere a copper suceptibility that makes this cells unable to thrive in the enviroment. We are also designing primers to built our new biosafety system based on mE genes and MgSO4, wich will be tested as soon as it is ready.
