BME PhD Prospectus Defense - Juan Jaramillo Montezco

Title: “Expansion of Biomolecular Sensor Functionality Using Artificial Cells and an Application in Water Contaminant Testing”

Advisory Committee: Erica Pratt, PhD – BU BME (Chair) Alexander Green, PhD – BU BME (Co-Advisor) Mark Grinstaff, PhD – BU BME, CHEM, MSE, MED (Co-Advisor) Aurelie Edwards, PhD – BU BME John Ngo, PhD – BU BME Paula Hammond, PhD – MIT ChemE

Abstract: Synthetic biology tools have become increasingly useful due to their ability to sense and respond to virtually any combination of physical, chemical, and biological inputs (flexibility) with low interference from their background (specificity). However, efforts to deploy these tools are often limited by living cells’ high degrees of complexity and randomness, which eventually lead to loss of system controllability and predictability and pose bio-contaminant risks if mishandled. Minimal synthetic cells, micron-size vesicles composed of the smallest number of genes necessary to sustain living functions, offer a convenient platform to engineer robust and stably deterministic biological outcomes. Efforts to study these platforms have yielded minimal cells able to (i) report on environmental factors using RNA-based biosensors, (ii) allow selective transport of small molecules across the cell wall using selective transport proteins, (iii) organize division rings necessary for reproduction, at specific locations within the cell wall, and (iv) produce adenosine triphosphate (ATP) as an energy source that can be used for downstream processes, to name a few.

In this work, we will engineer artificial cells, endowed with passive or active diffusion transmembrane transporters, which house environment-sensing riboswitches useful for water contaminant detection. We chose this application due to its severity: in 2019, deaths due to unsafe drinking water sources ranked #14 worldwide, ending the lives of 1.23 million people. Water contamination is a human and economic health crisis. Classical contaminants include heavy metals and pharmaceuticals, and emerging contaminants include per- and poly-fluoroalkyl substances (PFAS, i.e. “forever chemicals”). To date, deployment of rationally designed riboswitches against water contaminants has not been broadly realized, as sample preparation and removal of RNA degradants (e.g. RNAses), which abound in water samples, is nontrivial; testing of such samples would result in premature biosensor degradation and loss of signal. By encapsulating our riboswitches in artificial cells, we will protect them from environmental degradants. Moreover, we can reconstitute transport proteins across the lipid bilayer, endowing our biosensor with size or chemistry selectivity, and the ability to transport molecules uphill their chemical concentration gradient. As a result, we can expand riboswitches’ performance to lower analyte concentration ranges. Throughout Aim 1 of this work, we will rationally design and test an RNA-based aptaswitch sensor for heavy metal sensing. In Aim 2, we will optimize (a) the riboswitch encapsulation method and (b) the liposomal protein decoration method, which will allow us to demonstrate the expanded riboswitch performance. Finally, in Aim 3, we will demonstrate the generalizability of this biosensor design process by creating a broad PFAS detector.

Successful completion of this work will advance point-of-care testing of water contaminants, broaden RNA aptaswitch device functionality within complex water matrices, and allow these devices to detect contaminants at relevant, low concentration levels.