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Author

Ruhan Farsin

Date of Award

2019

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

Abstract

Sodalis glossinidius’s heme and heat stress survival mechanisms were studied by identifying functions of known heme induced genes, SG2427 and SG2061 and the heat shock genes, dnaK and grpE. Intron mutagenesis was used in an attempt to create three mutant strains of Sodalis with either SG2427, SG2061, or dnaK knocked out. With this technique, no viable ΔdnaK colonies formed, but ΔSG2427 and ΔSG2061 mutants, URSod26 and URSod28 were successfully created. URSod26 was then grown in varying concentrations of heme and measured for growth via optical density to analyze the potential of SG2427 to aid Sodalis’s growth in high heme conditions, but no difference in growth was observed compared to the wild-type (WT) strain, SodF/pAR1219. A similar assay was done measuring viable cells rather than optical density with URSod26 and URSod28 and using higher heme concentrations, revealing that while WT and URSod28 exhibit similar growth, URSod26 showed decreased growth at all heme concentrations. URSod26 was also inoculated in vivo in tsetse flies, via blood meals, but exhibited no growth and indeed died, implicating SG2427 as a necessary gene for Sodalis survival in its host. In regards to the thermal stress survival gene grpE, the Sodalis homologue of the gene was transformed into WT and ΔgrpE E. coli, and the strains were incubated at 30oC and 46oC in order to observe and compare its function to the E.coli homologue. Whereas ΔgrpE E. coli would not grow at 46oC prior to transformation, unlike the WT, the addition of Sodalis grpE restored growth in the mutant, suggesting the gene’s function is heavily conserved between the two species.

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