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.

Circadian expression characterization

We have already 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 already built) will be placed by means of Gibson assembly controlling the expression of this reporter.

New Biosafety strategies

We have already 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 susceptibility that makes this cells unable to thrive in the environment. We are also designing primers to built our new biosafety system based on mE genes and MgSO4, which will be tested as soon as it is ready.

Biolamp development

When all the lux genes are inserted into Synechocystis under the promoters recomended by our modelling we will make a new bioluminescense assay in the luminometer to test if our strain is ready to be grown in the biolamp. The biolamp prototype will be built so bacteria can be placed inside it. Finally we will characterize the sustainability of wt Synechocystis and our recombinant strain inside the biolamp.