Matthew Lebovich – Lauren Andrews Research Group
A Toolkit For Reprogramming Gut Microbiota Using Synthetic Genetic Circuits
Matthew Lebovich and Lauren Andrews
Through the implementation of designable genetic circuits, probiotic strains of bacteria can be used as non-invasive diagnostic tools for the gastrointestinal tract. In order for these programmed cells to be able to detect and report disease biomarkers after exiting the gut, the genetic circuits need to be able to record these signals it received within the gut environment using genetically- encoded memory. Complex memory register circuits would allow for multiplex interrogation and detection of disease biomarkers. We have developed a computational approach for the scalable design of genetic circuits that contain memory, which are known as sequential logic circuits. Importantly, these sequential logic circuits can also be implemented for temporal programming of cells. The theory-based approach to design sequential circuits from simple NOT gate responses is robust and makes accurate predictions for standard cell growth conditions. However, the way in which circuit component performance varies for different bacterial strains and gut- relevant environments is poorly understood and could lead to loss of performance of the circuit. Here we aim to develop a computational approach for the design of robust sequential logic circuits for in vivo diagnostic and therapeutic applications. In this work, we use a TetR family of repressors to build NOT and NOR logic gates that can be composed into complex sequential circuits. The NOT gates were characterized in the probiotic strain Escherichia coli Nissle 1917. Using this data, we designed and predicted the behavior of larger circuit designs. We present a set of genetic circuits that encode combinational logic and sequential logic and show that the circuit outputs are in close agreement with our quantitative predictions from the design algorithm.
Weiyue Xin – Maria Santore Research Group
Attractive and Repulsive Interactions between Plate-like Solid Membrane Domains in Phospholipid Membranes
This project employed giant unilamellar phospholipid vesicles to study the interactions between solid membrane domains which act more generally as plate- like inclusions, and it made comparison to a continuum model. The positions of multiple solid microscale membrane domains within single vesicles suggested the presence of an attractive minimum between domain pairs, short range interdomain repulsions, and long-range inter-domain attractions. We quantified the range of the potential minimum in vesicles containing only two domains, reporting features of a true pair potential. These findings contrast to the well-known repulsive interactions in vesicles containing phase separated fluid domains, or the purely attractive interactions of nanoscopic inclusions. As previous work has only reported repulsive interactions or attractive interactions individually, our discovery of a distinct minima translates to preferred domain positioning. These interactions result from the shear elasticity of the solid domains, beyond simply enhanced bending stiffness and shear rigidity, solid domains tend to expel Gaussian curvature into the fluid membrane phase, which generically competes with the global spherical topology of the vesicle. We also discovered the ability to toggle interactions, and shift the targeted positioning, by osmotic adjustment or mechanical manipulation of the fluid membrane by manipulating the ratio of membrane area to vesicle volume and employing micropipettes or osmotic pressure.
(This work is collaborated with Dr. Hao Wu and Prof. Gregory Grason from Polymer Science and Engineering Department at UMass Amherst.)