IGEM:Caltech/2007/Project Overview/Cro

Background
The lambda phage’s Cro is a 66 amino acid long protein that exists primarily as dimmers in the cell. Cro and lambda repressor can both potentially bind to three operator sites, OR1, OR2, and OR3.

Potential Binding Combinations
When repressor binds to OR2 [only], RNA polymerase bind to PRM and prevents its binding to the PR region, leading to transcription of cl tenfold (Positive Control) and turning off the cro gene (Negative Control). When repressor binds to OR1, RNA polymerase favors neither PRM nor PR. Polymerase access to PR is blocked, but the absence of repressor fails to attract polymerase to the PRM region, leading to a very low level of PRM. A repressor bound to OR3 blocks PRM transcription, but polymerase can still easily reach PR.

Lambda and e. coli life cycle
In a lysogenic cell, the repressor is usually bound to OR1 and OR2. Cooperativity between adjacent repressor molecules raises the operators’ affinities for repressor. A normal [lysogenic] repressor concentration causes transcription of cl and prevents cro synthesis. As repressor concentration increases further, repressor binds to OR3 and turns off the cl gene, allowing a constant level of repressor despite changes in growth rate. The switch to lysis, brought on by stimuli [such as ultraviolet light] damaging the bacteria’s DNA, an activated bacterial protein (RecA) starts cleaving repressor molecules, leading to a dropping repressor concentration. As OR1 and OR2 are now empty, cl transcription ceases, and polymerase binds to PR to transcribe cro. Cro binds independently to OR1, OR2, and OR3, preventing PRM transcription. The genes to the right of cro are also transcribes, which leads to the lytic phase.

Purpose
My task is to find the titration curve of the cro protein. Varying the Cro concentrations in bacteria (by changing amounts of ATC, which relieves the repression of the cro gene, and comparing to a standardized curve using the same ATC-relief promoter system driving GFP) and adding cro amber mutants (which are deficient in the production of Cro), and measuring percent lysis at each concentration, a [cro]-lysis curve can be generated, which would give upper and lower bounds for the amount of Cro that the riboregulator must “activate” in later integration phases of the project.