The Cro protein is a transcriptional regulator that plays an active role in the switch between the lysogenic and lytic viral cycles of bacteriophage lambda. Functional Cro proteins exist as dimers composed of two identical Cro subunits. It is suspected that the dimerization rate and the affinity of Cro’s subunits play a part in determining the activity of the protein in living cells. Synthetic genetic circuits have been constructed in order to determine the repression dynamics of Cro in vivo. These circuits are used to observe the ability of wild-type Cro and a pre-dimerized variant named scCro to modulate the expression of a reporter gene as compared to the unrepressed circuit. The unrepressed circuit that is used as a control is named the OPO circuit, and it contains no copy of the cro gene. The goal of this work was to construct an additional simple circuit with another variant cro gene (croF58W) in order to compare its in vivo regulatory dynamics in living cells with the binding dynamics that have been observed in a purified system. Wild-type Cro proteins have a phenylalanine at position fifty-eight, but CroF58W proteins have a tryptophan at this position. In vitro studies done using CroF58W variants have shown that CroF58W proteins are slower to dimerize but are thermodynamically more stable. Steady-state assays conducted in vivo with the CroF58W circuit showed an increased level of (3-galactosidase compared not only to the wild-type Cro and scCro circuits but also to our control OPO circuit
