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- All Subjects: Biomedical Engineering
- Creators: Brafman, David
- Resource Type: Text
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.
I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics.
Polymer drug delivery system offers a key to a glaring issue in modern administration routes of drugs and biologics. Poly(lactic-co-glycolic acid) (PLGA) can be used to encapsulate drugs and biologics and deliver them into the patient, which allows high local concentration (compared to current treatment methods), protection of the cargo from the bodily environment, and reduction in systemic side effects. This experiment used a single emulsion technique to encapsulate L-tyrosine in PLGA microparticles and UV spectrophotometry to analyze the drug release over a period of one week. The release assay found that for the tested samples, the released amount is distinct initially, but is about the same after 4 days, and they generally follow the same normalized percent released pattern. The experiment could continue with testing more samples, test the same samples for a longer duration, and look into higher w/w concentrations such as 20% or 50%.