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- All Subjects: Synthetic Biology
- Creators: Harrington Bioengineering Program
- Creators: Brafman, David
- 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.
Parkinson’s Disease (PD) is characterized by involuntary tremors, muscle rigidity, slowed movement, and cognitive impairments. Some people with PD experience “Freezing of Gait” (FOG), which is an acute inability to release effective stepping. The severity of Freezing of Gait can be influenced by disease duration, disease severity, and medication. Freezing of Gait can lead to an increased risk of falls, mood disorders, reduced quality of life, poorer cognition, and executive function impairments (Morris et. al.). It is important to understand how Freezing of Gait can affect cognition, as this can alter the plan of cognitive rehabilitation that the patient receives in order to improve their cognition. Within each of these domains, are a variety of tests that all claim to measure the same cognition the same way and achieve similar results. A recent meta analysis assessed this hypothesized effect. However, to best understand this relationship, it is important to assess whether the effects of FOG status on cognition are similar across cognitive tests of the same domain. This research was performed by using a previously completed meta analysis on PD tests and the domains that tests fall into. Each of the domains were analyzed individually, and the most common tests used within each domain were compared to determine their effect sizes. The effect sizes were then compared to determine which tests have a greater influence on cognition, and if the effect sizes of each test within the same domain are similar, showing that FOG affects cognition to a similar degree.