Date Written

Spring 4-19-2018

Document Type

Distinction Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biochemistry and Molecular Biology

Advisor

Dr. Jennifer Bennett

First Committee Member

Dr. Jennifer Bennett

Second Committee Member

Dr. John Tansey

Third Committee Member

Dr. David Robertson

Keywords

Streptomyces, cyclic di-GMP, bacteria, protein interactions

Subject Categories

Molecular Biology | Molecular Genetics

Abstract

Streptomyces is a genus of the phylum actinobacteria most commonly found as soil bacteria and used as a major source of antibiotics. RmdA and RmdB are phosphodiesterases that break down the ubiquitous second messenger cyclic-di-GMP which determines colony morphology and development of Streptomyces. The objective of this research is to identify whether RmdA will have interactions with itself using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) System. Each gene was fused into one of two BACTH vectors that encode a different domain of a single protein (T18 and T25) and then cotransformed into the BACTH indicator strain. The transformants were plated on the indicator plates, LB-X-Gal and MacConkey-maltose, and incubated to qualitatively show their possible interactions. If the proteins interact, they will bring the separated T18 and T25 domains in close proximity to produce beta-galactosidase on LB-X-Gal or ferment maltose on MacConkey-maltose which will be seen as blue or red colonies respectively. The plasmids containing T18-RmdA and T25-RmdA were successfully created and cotransformed to determine whether RmdA interacts with itself. When testing RmdA fused to T18 with an open C-terminus (T18-RmdA) and RmdA fused to T25 with an open C-terminus (T25-RmdA), no interactions were detected. This could be due to the T18 or T25 fragments blocking or preventing the function of the N-terminus of RmdA which could be required for protein interactions. Further testing is being conducted to determine if the N-terminus is needed to interact.

Available for download on Saturday, April 18, 2020

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