Research

pUtopia Project

The first project I worked on in the lab was the pUtopia project, which aimed at creating a cell line containing a landing pad for the integration of genes and gene circuits into the AAVS1 region of the mammalian genome. The landing pad is integrated through the use of CRISPR-Cas9 and homology driven repair into the AAVS1 region. During my introduction to research, I assisted in cloning, transfection, and microscopy for this project. I also performed PCR on genomic DNA to verify that the landing pad was correctly integrated into the AAVS1 region.

My first independent task was establishing a kill curve in order to determine the minimum inhibitory concentration of the drug 5-FC needed to kill cells that had integrated the incorrect region of the pUtopia plasmid, which contains the gene CodA. The drug 5-FC is used to negatively select against cells containing the CodA gene.

Degradation Tag

My first independent project in the lab involved a synthetic gene circuit known as the “linearizer”. The linearizer is a negative autoregulatory circuit in containing repressor gene TetR, which binds to its own promoter, and a downstream fluorescence marker (GFP). When exposed to doxycycline, expression of the fluorescence marker increases linearly with doxycycline dose.

My project was aimed at integrating a degradation tag, specifically the ODC degron, into this gene circuit, and assessing the changes in expression dynamics. Incorporating the ODC degron downstream of a specific gene will cause the protein to be expressed with a degradation tag,  thereby increasing its degradation time. I performed cloning to integrate the degradation tag downstream of the GFP gene. Using flow cytometry and microcopy, I comparted the GFP expression of HEK 293 cells containing the original linearizer, the linearizer with the ODC degron attached to the GFP protein, and the ODC degron attached to a TetR-GFP fusion protein. I analyzed the data and compared it to MATLAB models of the linearizers. I presented this research at the 2019 URECA poster symposium.

Toggle Switch

The project I am currently working on for my master’s thesis involves creating a genetic toggle switch from two preexisting linearizer circuits. By swapping the repressor genes between the two linearizers, the two circuits will repress each other, so that the expression of one repressor will repress the alternate repressor.

Einstein Summer 2019

During the summer of 2019, I participated in the SURP program at Albert Einstein College of Medicine, where I assisted in biochemistry research in the lab of Dr, Jonathan Lai. The aim of the research project with which I was involved was analysis of binding sites of broadly neutralizing antibodies onto the Zika virus envelope protein, with the ultimate goal of creating a vaccine target. Much of the research involved site directed mutagenesis of various regions within the Zika virus envelope gene. Mutated genes were then transfected into HEK cells, where they are expressed on the surface of the cells. HEK cells containing the Zika genes were then incubated with antibodies,  and flow cytometry was used to analyze the binding of antibodies to the cells. When a mutation prevented the antibody from binding to the cell, it is concluded that this mutation is in an epitope that is crucial for the antibody neutralization of the virus, and these epitopes are potential targets for therapies. I presented this research at a poster symposium at the conclusion of the summer.