Filtering by
- 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.
Ankle monitors are not the bright, kind alternative to incarceration that they are made to be. Advocates propose them as a solution to overcrowded carceral sites and excessive federal expenditure on public corrections agencies. Their logic being we can release certain incarcerated people to reduce prison, jail, and detention center populations and require them to pay for their monitoring to reduce prison expenditures. While there is potential for ankle monitors to achieve these aspirations, it is necessary to recognize how and where they can produce harm. Rather than being an alternative to incarceration, ankle monitors are a method of incarceration. They serve the same functions and hold the same power as prisons and jails with a more elusive form. In the current implementation of ankle monitors, we see individual bodies being transformed into sources of data to be capitalized upon by the government and private companies. Along with this, there is a shift of the financial burden of incarceration from prisons to the person being punished. This acts to further perpetuate the cycles of poverty and financial oppression that are seen within traditional forms of incarceration. Ankle monitor advocates also claim ankle monitors allow incarcerated people to enjoy freedom beyond prison walls and reintegrate into society. In reality, this is an oversimplification of freedom. Individuals with ankle monitors find themselves to be limited in their freedoms by restricted movement and stigmatization. They are unable to live a “normal” life because their ankle monitors prevent them from doing so. These people cannot move as they please, they cannot find and hold employment, and they cannot interact with people like they normally would. Ankle monitor usage must be critically examined and altered if it is to be considered a meaningful, gentle alternative to incarceration.