Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predicctions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research.
| Identifer | oai:union.ndltd.org:BGMYU2/oai:scholarsarchive.byu.edu:etd-7613 |
| Date | 01 December 2017 |
| Creators | White, Kylie MaKay |
| Publisher | BYU ScholarsArchive |
| Source Sets | Brigham Young University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | http://lib.byu.edu/about/copyright/ |
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