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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.
Pelvic Circumferential Compression Devices (PCCDs), an important medical device when caring for patients with pelvic fractures, play a crucial role in the stabilization and reduction of the fracture. During pelvic fracture cases, control of internal bleeding through access to the femoral artery is of utmost importance. Current designs of PCCDs do not allow vital access to this artery and in attempts to gain access, medical professionals and emergency care providers choose to cut into the PCCDs or place them in suboptimal positions with unknown downstream effects. We researched the effects on surface pressure and the overall pressure distribution created by the PCCDs when they are modified or placed incorrectly on the patient. In addition, we investigated the effects of those misuses on pelvic fracture reduction, a key parameter in stabilizing the patient during critical care. We hypothesized that incorrectly placing or modifying the PCCD will result in increased surface pressure and decreased fracture reduction. Our mannequin studies show that for SAM Sling and T-POD, surface pressure increases if a PCCD is incorrectly placed or modified, in support of our hypothesis. However, opposite results occurred for the Pelvic Binder, where the correctly placed PCCD had higher surface pressure when compared to the incorrectly placed or modified PCCD. Additionally, pressure distribution was significantly affected by the modification of the PCCDs. The cadaver lab measurements show that modifying or incorrectly placing the PCCDs significantly limits their ability to reduce the pelvic fracture. These results suggest that while modifying or incorrectly placing PCCDs allows access to the femoral artery, there are potentially dangerous effects to the patient including increased surface pressures and limited fracture reduction.
Pelvic Circumferential Compression Devices (PCCDs), an important medical device when caring for patients with pelvic fractures, play a crucial role in the stabilization and reduction of the fracture. During pelvic fracture cases, control of internal bleeding through access to the femoral artery is of utmost importance. Current designs of PCCDs do not allow vital access to this artery and in attempts to gain access, medical professionals and emergency care providers choose to cut into the PCCDs or place them in suboptimal positions with unknown downstream effects. We researched the effects on surface pressure and the overall pressure distribution created by the PCCDs when they are modified or placed incorrectly on the patient. In addition, we investigated the effects of those misuses on pelvic fracture reduction, a key parameter in stabilizing the patient during critical care. We hypothesized that incorrectly placing or modifying the PCCD will result in increased surface pressure and decreased fracture reduction. Our mannequin studies show that for SAM Sling and T-POD, surface pressure increases if a PCCD is incorrectly placed or modified, in support of our hypothesis. However, opposite results occurred for the Pelvic Binder, where the correctly placed PCCD had higher surface pressure when compared to the incorrectly placed or modified PCCD. Additionally, pressure distribution was significantly affected by the modification of the PCCDs. The cadaver lab measurements show that modifying or incorrectly placing the PCCDs significantly limits their ability to reduce the pelvic fracture. These results suggest that while modifying or incorrectly placing PCCDs allows access to the femoral artery, there are potentially dangerous effects to the patient including increased surface pressures and limited fracture reduction.