Date of Award
The Cro protein plays a critical role in the genetic switch, which governs the life cycle of the lysogenic bacteriophage lambda. Cro is a slow folding protein. The aim of this thesis is to test the idea that the folding rate of Cro has a direct effect on the dynamics of gene regulation in living cells. One way to test this hypothesis is to increase the rate of folding inside cells. SlyD is a peptidylprolyl isomerase that increases the rate of Cro folding in vitro. Strains of E.coli with varying levels of SlyD were infected with the lambda virus and the lysogenization frequency was determined for each. Although different protocols gave different probabilities, the frequency of lysogenization was significantly altered in SlyD deletion and SlyD overexpression strains. The preliminary conclusion from the experiment is that SlyD can enhance the fimction of Cro. Several Cro variants have been constructed that differ in their folding rates in vitro. A second test of the coupling between protein folding and gene regulation involves the construction of a simplified control circuit to measure the time required for Cro activity to appear after the induction of transcription. The circuit consists of the cro and lac Z gene, which are controlled by both the lac and lambda operators. Transcription that is initiated by induction of the lac repressor will continue until the accumulation of active Cro repressor in sufficient quantities to repress its own synthesis and the synthesis of p-galactosidase. IV We have begun the construction of this hybrid control sequence. Our results indicate that the plasmid that we now have does not contain the correct sequence of genes for our studies, and so further work will be necessary to obtain the desired product in order to move forward with the main goal of this study. 1 i V
Onwubiko, Chinwendu Chioma, "Studies on the Role of Foldign of the Cro Protein in the Genetic Switch of the Bacteriophage Lambda" (2003). Honors Theses. 2084.