Question:

Should we separate plasmids from Biobricks? Should each section apply to each construct?

Contents 1 Construction of plasmids 1.1 pSB1C3_IntK 1.2 pSB1A3_IntC (From 2010 Utah's iGEM team) 1.3 pSB1C3_IntK 1.4 Corroborating sequences 1.5 Conclusions 2 Transforming Synechocystis PCC 6803 2.1 Transformation 2.2 Reestreaking 2.3 Confirming DNA 2.4 Conclusions 3 Revealing phenotypes 3.1 Microscopy 3.2 Luminometer readings 3.3 Conclusions

Construction of plasmids

pSB1C3_IntK

Our first attempts to build the construct ( BBa_K743004) through Gibson Assemly were unsuccessful due to problems involving high compexity of the reaction, however we were able to Biobrick the recombination sites ( Part:BBa_K743000 and Part:BBa_K743001). Afterwards we decided to build a simple backbone first before continuing with more complex assemblies.

Through standard assembly we managed to build K743006 which led us to continue assembling our constructs through simpler Gibson Assemblies. In short time we were able to build our final constructs for the luciferase using 2 different versions of it available in the registry ( From Photorhabdus luminiscent, Part:BBa_K743014 and from Vibrio fisherii, Part:BBa_K743015) under an endogenous Synechocystis's promoter (transaldolase Reference???)

All constructs and parts have been verified by digestion and corroborated by sequencing.

pSB1A3_IntC (From 2010 Utah's iGEM team)

We decided to use this plasmid to place the LuxCDEG part of the Lux operon in this plasmid, however after various attempts to transform Synechocystis without success we reconsidered (see below SOMEWHERE!!! [[]]) due to problems regarding the design of this plasmid backbone (see below ANOTHERWHERE)

pSB1C3_IntK

When we reconsidered about using pSB1A3_IntC we designed a new plasmid backbone to place the LuxCDEG (substrate regeneration part of the Lux operon) under the Synechocystis

Corroborating sequences

Conclusions

Transforming Synechocystis PCC 6803

Transformation

Reestreaking

Confirming DNA

Conclusions

Revealing phenotypes

Microscopy

Luminometer readings

Conclusions