College of Integrative Sciences

The biofilm adhesion protein (Bap1) is a structural component of the Vibrio cholerae biofilm, a sticky mixture of proteins and glycans that help bacteria colonies survive hostile conditions while also contributing to virulence. Our lab discovered a unique 57-amino acid loop within the Bap1 protein necessary for stickiness to abiotic and biotic surfaces. This sticky peptide has the potential to be used in medical and industrial settings, similar to a mussel-inspired glue currently used as a tissue adhesive and industrial glue. The advantage of our system is that since it is simpler than the mussel protein; it might be more easily industrially produced and optimized.

The proposed research project involves the cloning and expression of the 57-amino acid peptide in
Escherichia coli. The goal is to optimize expression of the peptide, understand the mechanism for its
stickiness, and engineer adhesives inspired from it.

The research project integrates molecular biology (DNA editing), protein purification  (biochemistry/biophysics), and material science. He is working with Associate Professor Rich Olson (MB&B). 

Nakial is compiling a comprehensive review of the biosynthesis of the LPS core with Professor Erika Taylor (Chemistry) and Assistant Professor Phil Arevalo (Biology). 

Similar to how we as humans, have our defense mechanisms such as T-cells and macrophages, restriction enzymes are the classic defense mechanism for bacteria. They are able to cleave DNA at specific sequences to defend against an attacker, such as a phage. There is a large diversity among bacteria genetically where different restriction enzymes are expressed by different bacterial strains that balance between their own genes and invading bacteriophages. Many scientists have tried to look into restriction enzymes in terms of how they move around DNA and how it works within our system generally. The problem is that the mechanistic understanding of how REases bind and cleave DNA is very limited, which will become clearer with my continued research in the Etson Lab on the MvaI restriction endonuclease.

Restriction endonucleases are enzymes that are able to cleave duplex DNA at or near a specific nucleotide sequence. MvaI is a Type IIP restriction endonuclease found in Kocuria varians. Most Type IIP restriction endonucleases are homodimers that cleave a palindromic DNA sequence, however, MvaI has been shown to bind and cleave the pseudo palindromic sequence CC/WGG (where W can be A or T) as a monomer. Previous studies of the related monomeric restriction endonuclease BcnI revealed that this enzyme cleaves both strands of its pseudo palindromic sequence during a single binding event by flipping on the DNA. This kind of reorientation may require the protein to enter a pseudo-bound state, which can be disrupted by high salt concentrations. Alternatively, the protein may maintain nonspecific contacts with the DNA backbone, rendering the transition insensitive to ionic strength. Preliminary work with BcnI suggests that it does not pass through a pseudo-bound state. We are now investigating whether the same is true for MvaI. Our method uses a total internal reflection fluorescence microscopy (TIRF) based single molecule assay to observe DNA cleavage and the use of laminar flow cell devices that will help determine how salt impacts the reaction mechanism. This summer, we implemented NaCl as the salt in our experimental buffers. Research is still ongoing, but the data collected so far tells us that MvaI has no pseudo-bound state, and currently are exploring MvaI with the use of an acetate buffer. We also use quantum-dot labeled DNA substrates that are designed to report on either duplex cleavage or nicking of a specific strand of the pseudo palindromic sequence to determine whether there is a bias in initial binding orientation. We are able to set up a platform for TIRF microscopy and assure the binding of luminous nanoparticles, both of which are crucial to the success of our research, owing to the application of physics. Also, by using MATLAB's computer programming capabilities, we are able to examine our results and, ideally, understand the mechanism of single-molecule catalysis and bimolecular interactions. Nevertheless, our overall goal is to establish a model for how monomeric Type IIP restriction endonucleases bind and cleave duplex DNA.

1. The experimental side of designing a broadband limiter using ceramic resonators in the microwave regime. I code a robotic arm and a vector network analyzer to take measurements of specific structures as well as creating a simulation of the same setup in comsol.
2. Assisting on the experimental efforts of a project designed to find a coherent perfect absorption regime in a tetrahedron graph with a nonlinear resonator.
3. Assisting on the experimental efforts of a project designed to find a scar-state in a tetrahedron graph with a single variable bond length aimed at manipulating the phase of waves in the structure.