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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.
The distinctions between the neural resources supporting speech and music comprehension have long been studied using contexts like aphasia and amusia, and neuroimaging in control subjects. While many models have emerged to describe the different networks uniquely recruited in response to speech and music stimuli, there are still many questions, especially regarding left-hemispheric strokes that disrupt typical speech-processing brain networks, and how musical training might affect the brain networks recruited for speech after a stroke. Thus, our study aims to explore some questions related to the above topics. We collected task-based functional MRI data from 12 subjects who previously experienced a left-hemispheric stroke. Subjects listened to blocks of spoken sentences and novel piano melodies during scanning to examine the differences in brain activations in response to speech and music. We hypothesized that speech stimuli would activate right frontal regions, and music stimuli would activate the right superior temporal regions more than speech (both findings not seen in previous studies of control subjects), as a result of functional changes in the brain, following the left-hemispheric stroke and particularly the loss of functionality in the left temporal lobe. We also hypothesized that the music stimuli would cause a stronger activation in right temporal cortex for participants who have had musical training than those who have not. Our results indicate that speech stimuli compared to rest activated the anterior superior temporal gyrus bilaterally and activated the right inferior frontal lobe. Music stimuli compared to rest did not activate the brain bilaterally, but rather only activated the right middle temporal gyrus. When the group analysis was performed with music experience as a covariate, we found that musical training did not affect activations to music stimuli specifically, but there was greater right hemisphere activation in several regions in response to speech stimuli as a function of more years of musical training. The results of the study agree with our hypotheses regarding the functional changes in the brain, but they conflict with our hypothesis about musical expertise. Overall, the study has generated interesting starting points for further explorations of how musical neural resources may be recruited for speech processing after damage to typical language networks.