Our lab discovered that approximately 1 in 10,000 Escherichia coli cells in stationary phase remain in suspension after a high g-force centrifuge event. To establish the mechanism behind this curious phenotype, multiple mutant strains of E. coli were independently evolved such that the majority of their populations resisted migration when exposed to high centrifugal forces. Genomic DNA sequencing of the mutants' revealed unique, isolated mutations in genes involved in capsule synthesis and exopolysaccharide (EPS) production. Each mutant exhibits a novel mechanism that allows them to remain in suspension. The mutants were further characterized by determining their growth rates, strengths of resistance to various centrifugal forces, the phenotype's dependence on a carbon source, and timing of the phenotype's presentation. The results revealed: comparable mutant generation times to the wild-type strain, variable resistance to centrifugal force, phenotype dependence on carbon source, and phenotype presentation during early stationary phase. To interrogate the mechanism by which these cells stay in suspension the production of EPS was quantified, and gene knock-outs were performed. Quantification of the EPS revealed approximately a seventeen-fold increase in EPS in the mutants' compared to the wild-type strain. Gene knock-outs revealed the EPS produced can be attached to the outer-membrane or freely secreted into the media by different mechanisms. In addition, this mechanism was further confirmed to be responsible for the centrifuge resistant trait by attaching extracted EPS to polystyrene microspheres. Experimental results show that mutant extracted EPS treated beads caused increased bead retention in suspension compared to wild-type EPS treated beads. These results reveal that E. coli is using a novel mechanism to adapt to a new environmental factor introduced to remove the bacteria. With the discovery of this mechanism and the transferability to inorganic objects industrial applications are now envisioned where particle sedimentation is controllable and mixtures remain homogenized by attaching optically transparent biomolecules.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-1290 |
| Date | 01 January 2018 |
| Creators | Kessler, Nickolas |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Honors Undergraduate Theses |
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