The human body’s circadian rhythm or “clock” regulates the daily cycles of many physiological and metabolic processes such as the sleep wake cycle and feeding rhythms. The biological processes and temporal coordination are crucial to the health and survival of organisms. The processes occur with the capacity to oscillate with a wide variety of periods that are controlled by the interplay of numerous molecular factors that orchestrate complex feedback loops and processes that are fundamentally mediated by gene expression and the events that follow. In humans, circadian rhythm arises from circadian complexity and involves the activity of several components, most importantly the negative elements period homologues PER1 PER2 and the CRY1 and CRY2 crytpochrome elements along with the positive acting proteins of CLOCKA and BMAL1. The figure below depicts the molecular interactions in mammalian circadian feedback loops. The oscillator is composed of interlocking feedback loops that regulate the abundance and activity of transcription factors. These transcription factors control the expression of genes in the output pathways from the oscillator, resulting in behavioral and physiological rhythms.

The CLOCK and BMAL1 form heterodimers and activate transcription of the Per and Cry genes that form the period. The critical mechanism in the pathway occurs when PER and CRY proteins bind as heterodimers and inhibit CLOCK and BMAL1 transcription via sequestration.

Our Project: The Sequestillator

We can subdivide our clock into two parts: the activator module and the repressor module. The activator module consists of the constitutive promoter driving the NifA gene, thereby producing a constant amount of NifA. We will be using three different BioBrick promoters - corresponding to low, medium, and high outputs of NifA respectively. The NifA protein binds to the nifHp promoter of the repressor module, activating transcription of the NifL gene. Once NifL dimerizes, it can bind to the NifA hexamer, hence preventing NifA from binding to NifHp. This sequestration effect provides the clock's negative feedback loop that is essential for oscillations. Our hope is to put these modules on the E.coli chromosome using a BioBrick compatible Arabinose Landing Pad (our iGEM 2007 project) and a Leucine Landing Pad (a construct of a former Ninfa lab member, Dong Eun Chang). We will use E.coli strain NCM 1971, which has the nifHp driving lacZ on the chromosome. This way, we can test the amounts of NifA via Beta-galactosidase activity and test the amount of NifL via fluorescent microscopy.