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- All Subjects: Synthetic Biology
- Creators: Brafman, David
- Creators: Harrington Bioengineering Program
- Status: Published
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.
Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
Objective: To determine if patients’ insurance status or the income level of their zip code of residence affect their quality of life or overall survival after enrollment in clinical trials for cancer treatment. Methods: Data were collected from cancer treatment trials conducted through the North Central Cancer Treatment Group and the Alliance for Clinical Trials in Oncology. 700 subjects with baseline quality of life scores were analyzed to explore potential differences in quality of life indicators by insurance group. 624 patients with valid US zip codes were also analyzed based on the median household income of their zip code to determine any associations with quality of life. Overall survival was also analyzed by insurance group and by income quartile. Results: 700 subjects (mean age 59 years, 53% male) were included. 49% had private insurance only, 30% had public insurance only, 8.9% had both private and public insurance, 1.4% had no insurance, and 10% had other insurance. 13% of patients came from zip codes in the bottom quartile by median income, 20% came from the second quartile, 25% from the third quartile and 42% from the top quartile. No significant differences were found in baseline quality of life scores between insurance groups or income quartiles. Patients with both private and public insurance had higher baseline fatigue scores compared to only private, only public, or other insurance. No significant difference was found in baseline fatigue scores by income quartile. No significant differences were found in overall survival by insurance group or income quartile. Conclusions: Patients with both private and public insurance may need more extensive interventions than patients with other insurance statuses due to their higher baseline fatigue scores. Future studies are needed to further investigate the effects of neighborhood advantage level on quality of life indicators.