|Matthew Lebovich, 2nd Year
Program: Chemical Engineering
Advisor: Lauren Andrews, PhD.
Education: Rensselaer Polytechnic Institute, Chemical Engineering, BS 2015
Certain commercially available probiotic strains such as L. acidophilus and E. coli Nissle 1917 have proved beneficial in managing inflammatory bowel diseases. Modification and tighter control of their functions through genetic engineering has the potential to increase the therapeutic effects of such strains as well as allow them to perform as diagnostic tools and drug delivery agents. Through the implementation of genetic circuits an engineered organism can be genetically programmed to sense specific stimuli and respond according to its design. Once a thorough understanding of how circuit parts behave is achieved, software developed in other labs that can be used to automate the design of complex circuits and predict their behavior. However, most work has been done only in lab strains and under laboratory conditions. The way in which circuit performance is affected by environmental factors is poorly characterized and can lead to various problems and performance issues. Genetic circuits operate using the same concepts as electrical ones. Logic gates are assembled together and determine an on or off output signal based on an input. In biology though, signals are not binary. Certain systems do provide us with distinct on and off states though, based on the level of input. The level of input that corresponds to a switching from an off to an on state can change however as conditions and circuit constructs vary. In order to incorporate genetic circuits into probiotic organisms, further part characterization is needed under various conditions so that circuits will perform as designed. My project aims to first characterize the behavior of a library of repressor based NOT logic gates with distinct on and off states, which can be arranged to create any circuit. They will be examined under lab conditions as well as those mimicking the human gut. Since different strains of bacteria do not behave the same, when moving from a lab strain of E.coli to a probiotic organism, parts will have to examined for their fidelity. The purpose of this is to determine how circuit performance might change under medically relevant conditions such as the low oxygen environment in the gut without antibiotic selection normally used in the lab. If the circuit parts behave differently in these conditions, then certain circuits may have to be tuned or reconfigured and new design criteria can be elucidated. Once simpler circuit parts are characterized, more complex circuits can be designed and built. Circuits that I am particularly interested in are multi input gates such as AND or NOR as well as multi input latches such as the data latch or set reset latch. Latch circuits are particularly interesting because they can be used as rewritable memory. After implementing and verifying the functionality of these circuits, this will allow a probiotic organism to record the presence of a signal or metabolite in the gut and retain that information once the signal is no longer present so that it can be analyzed later. In this way the organism can be used to display the presence of disease markers and possibly locally deliver a therapeutic in response only when the markers are present. Aside from just the medical field, genetic circuits can be applied to any field that needs dynamic control of microbes. After circuits have been tested and verified the next step will be to collaborate with a group experienced in mouse studies to evaluate if our circuit behavior in vivo matches that in vitro. From there different sensors and end products can be incorporated to change the circuit’s inputs and outputs and the efficacy of genetic circuits as diagnostic or therapeutic tools can then be examined.