Transcriptional programming in a Bacteroides consortium

Abstract

Bacteroides species are prominent members of the human gut microbiota. The prevalence and stability of Bacteroides in humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community of Bacteroides (consortium transcriptional programming) with genetic circuit compression. Circuit compression requires systematic pairing of engineered transcription factors with cognate regulatable promoters. In turn, we demonstrate the compression workflow by designing, building, and testing all fundamental two-input logic gates dependent on the inputs isopropyl-β-D-1-thiogalactopyranoside and D-ribose. We then deploy complete sets of logical operations in five human donor Bacteroides, with which we demonstrate sequential gain-of-function control in co-culture. Finally, we couple transcriptional programs with CRISPR interference to achieve loss-of-function regulation of endogenous genes—demonstrating complex control over community composition in co-culture. This work provides a powerful toolkit to program gene expression in Bacteroides for the development of bespoke therapeutic bacteria.

Performance Prediction of Fundamental Transcriptional Programs

“…An important feature of transcriptional programming is the ability to network repressors and antirepressors to build multiple-INPUT operations that are compressed. 28,30 In our current study, we accomplished this with the SE-PA architecture to form simple two-node networks of transcription factors binned via the alternate DNA binding function�e.g., YQR|O 1 , HQN|O ttg , or GKR|O gac . This SE-PA network form resulted in seven orthogonal (binned) DNA binding networks, with interbin communication facilitated via the INPUT signals (see Supporting Note 10).…”

“…Significant efforts have been devoted to engineering logical (decision-making) responses within a variety of chassis cells as a general proof-of-concept, − and for a variety of logic-based applications e.g. , biosensing, ,, biological clocks, − oscillators, ,− controllers, ,− and therapeutics. − An emerging technology in biotic decision-making is transcriptional programming. − Transcriptional programming makes use of fundamental logic principles by assigning an inducer molecule as the INPUT and by assigning a coupled regulated reading frame (coding or noncoding) as the OUTPUT. The operating constraints for said biotic programs are predicated on digitizing the INPUT to 0 or 1, where an INPUT 1 is achieved via the maintenance of saturating concentrations of the cognate inducer moleculetypically 10 mM.…”

“…, biosensing, 7 , 10 , 11 biological clocks, 12 − 15 oscillators, 12 , 16 − 19 controllers, 5 , 20 − 22 and therapeutics. 23 − 28 An emerging technology in biotic decision-making is transcriptional programming. 28 − 30 Transcriptional programming makes use of fundamental logic principles by assigning an inducer molecule as the INPUT and by assigning a coupled regulated reading frame (coding or noncoding) as the OUTPUT.…”

“… 23 − 28 An emerging technology in biotic decision-making is transcriptional programming. 28 − 30 Transcriptional programming makes use of fundamental logic principles by assigning an inducer molecule as the INPUT and by assigning a coupled regulated reading frame (coding or noncoding) as the OUTPUT. The operating constraints for said biotic programs are predicated on digitizing the INPUT to 0 or 1, where an INPUT 1 is achieved via the maintenance of saturating concentrations of the cognate inducer molecule—typically 10 mM.…”

Machine learning–aided real-time detection of keyhole pore generation in laser powder bed fusion

“…Their interplay often leads to structural defects, such as porosity. One common porosity is caused by the momentary collapse of the vapor depression zone, known as keyhole porosity (4)(5)(6)(7)(8).…”