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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.
SUMMARY: A failed attempt to conduct a systematic review of disparities in racial inclusivity in stroke rehabilitation research: A call to action Group Members: Adeline Beeler & Mikayla McNally Faculty Mentor(s): Dr. Sydney Schaefer & Dr. Keith Lohse Topic Overview: Stroke is responsible for the death of an individual every four minutes in the United States. While all Americans are gravely affected by this statistic, Black Americans are at a significantly increased risk of first stroke incidence when compared to their white counterparts, majorly due to heightened prevalence of stroke risk factors. Not only does race contribute as a factor in stroke incidence, but it also has a considerable impact in the physical impairment of Black Americans following stroke occurrence. While it still remains unclear as to whether or not stroke plays a significant role in stroke rehabilitation efforts, there is a clearly demonstrated need for increased reporting or participation of Black Americans in stroke rehabilitation clinical trials to have the ability to conduct a systematic review of these racial disparities in the near future. In the analysis of 36 stroke rehabilitation-related clinical research studies, 80% of selected trials failed to report any participant racial demographics, with 77.3% of the NIH-funded trials not reporting, as well. Out of the 7 trials that did provide some sort of participant racial information, only 5 successfully provided statistically significant racial data compared to the remainder that simply categorized participants’ race as “white” or “other.” In order to fully investigate the effects of race on stroke rehabilitation, it is imperative that researchers collect and report equally distributed and diverse participant racial data when publishing clinical research. Potential methods of improvement for researchers to include more racially diverse subject populations include more comprehensive and in-depth advertising and recruitment strategies for their studies. Research Methods: In order to produce accurate analyses of the current state of the relationship between race and stroke rehabilitation efforts, 36 stroke rehabilitation clinical research trials from various locations across the United States were identified using the Centralized Open-Access Rehabilitation Database for Stroke (SCOAR). These trials were evaluated in order to extract relevant data, such as number of trial participants, average age of participants, if the research trial was funded by the National Institute of Health (NIH) or not, and any reported participant racial demographic details. Trends across these categories were compared between all trials to determine if any disparities existed in providing data sufficient to support the relationship between varying racial populations and stroke rehabilitation efforts. Future Project Efforts: Future efforts will include the completion of submitting a Point of View/Directions for Research article for publication to offer an opportunity for clinical and basic researchers to examine the discrepancies surrounding racial inclusivity in stroke rehabilitation clinical research. The aim is to improve the ability of clinicians to interpret the literature, translate research studies into practices, and better direct future experiments. Further identification of stroke rehabilitation clinical research trials will be necessary, as well as modifications to current written work content.
SUMMARY: A failed attempt to conduct a systematic review of disparities in racial inclusivity in stroke rehabilitation research: A call to action Group Members: Adeline Beeler & Mikayla McNally Faculty Mentor(s): Dr. Sydney Schaefer & Dr. Keith Lohse Topic Overview: Stroke is responsible for the death of an individual every four minutes in the United States. While all Americans are gravely affected by this statistic, Black Americans are at a significantly increased risk of first stroke incidence when compared to their white counterparts, majorly due to heightened prevalence of stroke risk factors. Not only does race contribute as a factor in stroke incidence, but it also has a considerable impact in the physical impairment of Black Americans following stroke occurrence. While it still remains unclear as to whether or not stroke plays a significant role in stroke rehabilitation efforts, there is a clearly demonstrated need for increased reporting or participation of Black Americans in stroke rehabilitation clinical trials to have the ability to conduct a systematic review of these racial disparities in the near future. In the analysis of 36 stroke rehabilitation-related clinical research studies, 80% of selected trials failed to report any participant racial demographics, with 77.3% of the NIH-funded trials not reporting, as well. Out of the 7 trials that did provide some sort of participant racial information, only 5 successfully provided statistically significant racial data compared to the remainder that simply categorized participants’ race as “white” or “other.” In order to fully investigate the effects of race on stroke rehabilitation, it is imperative that researchers collect and report equally distributed and diverse participant racial data when publishing clinical research. Potential methods of improvement for researchers to include more racially diverse subject populations include more comprehensive and in-depth advertising and recruitment strategies for their studies. Research Methods: In order to produce accurate analyses of the current state of the relationship between race and stroke rehabilitation efforts, 36 stroke rehabilitation clinical research trials from various locations across the United States were identified using the Centralized Open-Access Rehabilitation Database for Stroke (SCOAR). These trials were evaluated in order to extract relevant data, such as number of trial participants, average age of participants, if the research trial was funded by the National Institute of Health (NIH) or not, and any reported participant racial demographic details. Trends across these categories were compared between all trials to determine if any disparities existed in providing data sufficient to support the relationship between varying racial populations and stroke rehabilitation efforts. Future Project Efforts: Future efforts will include the completion of submitting a Point of View/Directions for Research article for publication to offer an opportunity for clinical and basic researchers to examine the discrepancies surrounding racial inclusivity in stroke rehabilitation clinical research. The aim is to improve the ability of clinicians to interpret the literature, translate research studies into practices, and better direct future experiments. Further identification of stroke rehabilitation clinical research trials will be necessary, as well as modifications to current written work content.