Overview

This year, we're focusing on the LacI, TetR, and cI Lambda repressors and their respective promoters. Our goal is to obtain a variety of behaviors from each of these parts by changing their sequences randomly, or mutating them. With this range of behaviors, we'll be able to design systems to exhibit targeted behavior depending on the chosen repressor. This will be a valuable contribution to the registry which contains many parts that have a limited range of activities such as always strongly on or inducible. Having a well-characterized range of activity of a single part will provide more opportunities to fine tune a system with an added layer or regulation. When coupled with another part that has a similar range of activity, a repressible promoter with a repressor for example, there is a combinatorial effect which gives the user even more control.

General Construct

LacI Repressor

The lac repressor is responsible for regulating the metabolism of lactose. In the absence of lactose, LacI forms a tetramer with identical subunits which appears as two dimers. Each dimer binds in the major groove of the DNA binding region which subsequently blocks the RNA polymerase from binding. In nature, allolactose will bind the repressor leading to transcription of the lac operon. Using IPTG as an inducer has the same effect as allolactose. Read more about it here.

Tet Repressor

E. coli have evolved a resistance to the tetracycline antibiotic which is found in the tetracycline operon. This operon is regulated by TetR, the tetracycline repressor, which is a dimeric protein that binds the tetracycline repressible promoter. We mutated both of these. Read more about it here.

cI Lambda

This repressor originates from Lambda phage. Having either lytic or lysogenic life cycles, this bacteriophage infects it's E. coli host with double stranded DNA. cI binds at OR1, OR2 and OR3 sites with preference given to the OR1 site. For more information on how we mutated this repressor and it's associated promoter, click here.