Enlarging the synthetic biology toolbox for Saccharomyces cerevisiae A new synthetic reporter system for transcription dynamics analysis

Abstract

Green fluorescent protein (GFP) has been broadly used as an efficient reporter system, but not only its stability makes it unsuitable for monitoring transcription dynamics but also the oxygen-demanding chromophore formation restricts its application in aerobic systems. An ubiquitin fusion strategy and an N-degron including a destabilizing N-terminal residue and lysine containing Δk linker can tune the decay rate and provide a range of different stabilities, which is theoretically available for any protein. By N-terminally fusing ubiquitin and N-degron with the GFP-like TurboGFP and a novel flavin mononucleotide (FMN) binding fluorescent protein (FbFP), which works both in aerobic and anaerobic conditions, new destabilized reporter proteins were synthesized and evaluated at transcription level and functional translation level. With methionine, glutamic acid and tyrosine corresponding to relatively strong, middle and weak stability at the conjugate of ubiquitin and Δk linker, TurboGFP and FbFP were integrated into the chromosome of Saccharomyces cerevisiae under control of the inducible GAL1 promoter. The transcription level in each strain was quantified by RT-qPCR and as expected while the functional translation level, i.e. fluorescence intensity was very low. The destabilizing modifications were suspected to affect the fluorescence intensity. Therefore FbFP constructs containing different parts of the modification were developed and proved that the N-terminally fused ubiquitin and linker significantly affected the fluorescence although the mechanisms behind this require further study.