HTGAA Final Project Proposal Cell-Free Butyrate Biosensor for Gut Health Diagnostics Author: Emmanuel Pereyra, Rosario, ARG
Date: May 2026
System: Cell-Free Expression (BL21 DE3 Lysate)
Industry Partners: Twist Bioscience · Ginkgo Bioworks · Opentrons
SECTION 1: ABSTRACT Short-chain fatty acids (SCFAs), particularly butyrate, are critical metabolites produced by gut microbiota through fermentation of dietary fiber and serve as key indicators of gut microbiome health and colonocyte function. Dysregulation of butyrate levels has been implicated in inflammatory bowel disease (IBD), colorectal cancer (CRC), and broader metabolic dysfunction, yet current clinical detection methods rely on expensive, slow, and laboratory-intensive chromatographic techniques inaccessible to most clinical settings. This project proposes the design and experimental validation of a cell-free transcription factor-based biosensor capable of detecting butyrate in stool sample extracts with high sensitivity and specificity. The central hypothesis is that a synthetic genetic circuit encoding a butyrate-responsive BudR transcription factor coupled to a NanoLuc luciferase reporter can be expressed in a BL21 DE3 cell-free lysate system to produce a quantitative, dose-dependent luminescent signal in response to physiologically relevant butyrate concentrations. Aim 1 will design, synthesize, and functionally validate the BudR-NanoLuc biosensor construct ordered from Twist Bioscience and tested in an automated 96-well cell-free expression platform at Ginkgo Bioworks. Aim 2 will optimize biosensor sensitivity, dynamic range, and matrix compatibility with stool extracts. Aim 3 envisions deployment of this biosensor technology as a portable, point-of-care diagnostic platform for gut microbiome health monitoring applicable to IBD screening and microbiome research. This project integrates synthetic biology, cell-free expression, automated liquid handling, and clinical diagnostic design into a cohesive and immediately actionable research program.