(TetR) operator sequences. Second, to reduce the disparity between the lifetimes of the repressor proteins and the mRNAs, a carboxy terminal tag based on the ssrA-RNA sequence was added at the 3' end of each repressor gene. This tag is recognized by proteases which target the protein for degradation. The design was implemented using a low-copy plasmid encoding the repressilator and a higher-copy reporter, which were used to transform a culture of E. coli. Naturally Occurring Repressilators Plants Circadian circuits in plants feature a transcriptional regulatory feedback loop called the repressilator. In the core oscillator loop (outlined in gray) in A. thaliana, light is first sensed by two cryptochromes and five phytochromes. Two transcription factors, Circadian Clock Associated 1 (CCA1) and Late Elongated Hypocotyl (LHY), repress genes associated with evening expression like Timing of CAB expression 1 (TOC1) and activate genes associated with morning expression by binding to their promoters. TOC1, an evening gene, positively regulates CCA1 and LHY via an unknown mechanism. Evening-phased transcription factor CCA1 Hiking Expedition (CHE) and histone demethylase jumonji C domain-containing 5 (JMJD5) directly repress CCA1. Other components have been found to be expressed throughout the day and either directly or indirectly inhibit or activate a consequent element in the circadian circuit, thereby creating a complex, robust and flexible network of feedback loops. Morning-Phase Expression The morning-phase expression loop refers to the genes and proteins that regulate rhythms during the day in A. thaliana. The two main genes are LHY and CCA1, which encode LHY and CCA1 transcription factors. These proteins form heterodimers that enter the nucleus and bind to the TOC1 gene promoter, repressing the production of TOC1 protein. When TOC1 protein is expressed, it serves to regulate LHY and CCA1 by inhibition of their transcription. This was later supported in 2012 by Dr. Alexandra Pokhilo, who used computational analyses to show that TOC1 served this role as an inhibitor of LHY and CCA1 expression. The morning loop serves to inhibit hypocotyl elongation, in contrast with the evening-phase loop which promotes hypocotyl elongation. The morning phase loop has shown to be incapable of supporting circadian oscillation when evening-phase expression genes have been mutated, suggesting the interdependency of each component in this naturally-occurring repressilator. Evening-Phase Expression Early Flowering 3 (ELF3), Early Flowering 4 (ELF4) and Phytoclock1 (LUX) are the key elements in evening-phased clock gene expression in A. thaliana. They form the evening complex, in