Dynamics of Transcriptional Repression by λ Cro Repressor In Vivo

Author

Dustin Land Markle

Date of Award

2007

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Michael Mossing

Relational Format

Dissertation/Thesis

Abstract

Cro is a regulatory protein that participates in the life cycle switch of bacteriophage lambda (X). While wild-type Cro is produced as a single domain polypeptide monomer, two monomers must assemble to form a dimer before Cro can act as a functional transcriptional repressor. We suspect that dimerization of Cro monomers determines, in part, the dynamics of the activity of the protein in vivo. Rate and equilibrium constants for dimerization of several Cro variants have already been determined in vitro,, and dimerization of Cro monomers is slow relative to other similar proteins. This study is concerned with exploring the in vivo dimerization rates of Cro variants. Synthetic genetic circuits have been constructed and integrated into the genome of a strain of Escherichia coli to allow induction and repression of the expression of Cro protein variants. This allows for study of the dimerization process and the dynamics of subsequent repression of the circuit. Our strains contain either wild-type Cro or a synthetic variant, single-chain Cro (scCro), which does not require dimerization to function as a repressor. Also integrated into our strains is the lacZ gene for pgalactosidase, our reporter enzyme. The experimental rationale is to induce in separate strains the production of our two Cro variants, along with P-galactosidase, and then to compare the kinetics of IV repression of the circuits by the Cro variants to determine the effect of dimerization in vivo. A majority of our work has consisted of combining and optimizing several pre¬ existing methods of p-galactosidase assays to fit our particular experimental conditions. Our assays have been completed for both short time-course and long-term levels of induction and show more rapid transcriptional repression by our scCro variant compared with wild-type Cro. These results support the proposition that dimerization plays a role in repression dynamics. V

Recommended Citation

Markle, Dustin Land, "Dynamics of Transcriptional Repression by λ Cro Repressor In Vivo" (2007). Honors Theses. 2059.
https://egrove.olemiss.edu/hon_thesis/2059

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