Transcript Barcoding Illuminates the Expression Level of Synthetic Constructs in E. coli Nissle Residing in the Mammalian Gut.

The development of robust engineered probiotic therapies demands accurate knowledge of genetic construct expression in the gut. However, the monetary and ethical costs of testing engineered strains in vertebrate hosts are incompatible with current high-throughput design-build-test cycles. To enable parallel measurement of multiple construct designs, we placed unique DNA barcodes in engineered transcripts and measured barcode abundances via sequencing. In standard curve experiments, the barcode sequences exhibited consistent relationships between input and measured abundances, which allowed us to use transcript barcoding to measure expression levels of 30 GFP-expressing strains of E. coli Nissle in parallel. Applying this technology in culture and in the mouse gut, we found that GFP expression in the gut could often be predicted from expression levels in culture, but several strains exhibited gut-specific expression. This work establishes the experimental design parameters and advantages of transcript barcoding to measure the performance of many engineered probiotic designs in mammalian hosts.