Date of Award

5-2015

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biology

Advisor/Committee Chair

Marlene Belfort

Committee Member

Daniel Wulff

Abstract

A gene consists of coding segments, exons, which are maintained and contribute to the final mRNA product, while non-coding segments, introns, are spliced out through RNA splicing mechanisms to code a functional gene. The mobility of the spliced introns and factors that affect mobility are an important field of study evolutionarily whereas splicing defects have medical implications. For this study, the genome of the Lactococcus lactis bacterial strain, IL1403 and the group II intron of interest, L1.LtrB were utilized. This intron moves into new sites on DNA by a process termed retrotransposition (RTP). Our goal is to identify genes that regulate retrotransposition of L1.LtrB in L. lactis. To do this, we are making a transposon mutant library in L. lactis strain IL1403. The pG+host::ISS1 plasmid was used to introduce the ISS1 transposon into the genome of IL1403 at random positions, creating a library of mutants. The intron donor plasmid, containing the Ll.LtrB intron was also introduced into IL1403 and later used for retrotransposition assays that will determine the degree by which certain genes affect Ll. LtrB intron mobility. We have also developed a method to screen mutants for RTP levels via hybridization and selective media. So far we have generated over 1000 mutants with several showing stimulatory or inhibitory effects on retrotransposition. Genetic recombineering will follow allowing recreation of mutants for further analysis. This will occur after high-throughput sequencing which will localize specific genes that will give clues as to which host facts increase or decrease retrotransposition.

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