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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.
The cocktail party effect describes the brain’s natural ability to attend to a specific voice or audio source in a crowded room. Researchers have recently attempted to recreate this ability in hearing aid design using brain signals from invasive electrocorticography electrodes. The present study aims to find neural signatures of auditory attention to achieve this same goal with noninvasive electroencephalographic (EEG) methods. Five human participants participated in an auditory attention task. Participants listened to a series of four syllables followed by a fifth syllable (probe syllable). Participants were instructed to indicate whether or not the probe syllable was one of the four syllables played immediately before the probe syllable. Trials of this task were separated into conditions of playing the syllables in silence (Signal) and in background noise (Signal With Noise), and both behavioral and EEG data were recorded. EEG signals were analyzed with event-related potential and time-frequency analysis methods. The behavioral data indicated that participants performed better on the task during the “Signal” condition, which aligns with the challenges demonstrated in the cocktail party effect. The EEG analysis showed that the alpha band’s (9-13 Hz) inter-trial coherence could potentially indicate characteristics of the attended speech signal. These preliminary results suggest that EEG time-frequency analysis has the potential to reveal the neural signatures of auditory attention, which may allow for the design of a noninvasive, EEG-based hearing aid.