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- All Subjects: Synthetic Biology
- All Subjects: Flexion
- 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.
Falls are known to be a common occurrence and a costly one as well, as they are the second leading cause of unintentional deaths and millions of other injuries worldwide. Falls often occur due to an increase in trunk flexion angle, so this experiment aims to reduce the trunk flexion received while stepping over an obstacle. To achieve this a soft actuator was attached to the trunk and pressure was sent as subjects walked and stepped over an obstacle presented on a treadmill. The pressure is meant to stiffen the back which should in theory reduce the trunk flexion angle and lower the chances of falling. In this experiment, two groups were tested: three participants from a control group (healthy young adults) and three participants from an experimental group (healthy elderly adults). Since elderly adults have the highest fall risk due to overall lack of stability, they are the experimental group and the focus for this experiment. The results from the study showed that elderly adults had a beneficial effect with the soft actuator as there was a noticeable difference in trunk flexion when the device was attached. The experiment also supported prior research that stated that trunk flexion was greater in elderly adults than younger adults. Despite the positive results, further studies should be done to prove that the soft devices influence lowering trunk flexion angle as well as to see if the device has any noticeable effect on younger adults.