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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.
Following a study conducted in 1991 supporting that kinesthetic information affects visual processing information when moving an arm in extrapersonal space, this research aims to suggest utilizing virtual-reality (VR) technology will lead to more accurate and faster data acquisition (Helms Tillery, et al.) [1]. The previous methods for conducting such research used ultrasonic systems of ultrasound emitters and microphones to track distance from the speed of sound. This method made the experimentation process long and spatial data difficult to synthesize. The purpose of this paper is to show the progress I have made in the efforts to capture spatial data using VR technology to enhance the previous research that has been done in the field of neuroscience. The experimental setup was completed using the Oculus Quest 2 VR headset and included hand controllers. The experiment simulation was created using Unity game engine to build a 3D VR world which can be used interactively with the Oculus. The result of this simulation allows the user to interact with a ball in the VR environment without seeing the body of the user. The VR simulation is able to be used in combination with real-time motion capture cameras to capture live spatial data of the user during trials, though spatial data from the VR environment has not been able to be collected.