An official website of the United States government
Abstract The tenets of intelligent biological systems are (i) scalable decision-making, (ii) inheritable memory, and (iii) communication. This study aims to increase the complexity of decision-making operations beyond standard Boolean logic, while minimizing the metabolic burden imposed on the chassis cell. To this end, we present a new platform technology for constructing genetic circuits with multiple OUTPUT gene control using fewer INPUTs relative to conventional genetic circuits. Inspired by principles from quantum computing, we engineered synthetic bidirectional promoters, regulated by synthetic transcription factors, to construct 1-INPUT, 2-OUTPUT logical operations—i.e. biological QUBIT and PAULI-X logic gates—designed as compressed genetic circuits. We then layered said gates to engineer additional quantum-inspired logical operations of increasing complexity—e.g. FEYNMAN and TOFFOLI gates. In addition, we engineered a 2-INPUT, 4-OUTPUT quantum operation to showcase the capacity to utilize the entire permutation INPUT space. Finally, we developed a recombinase-based memory operation to remap the truth table between two disparate logic gates—i.e. converting a QUBIT operation to an antithetical PAULI-X operation in situ. This study introduces a novel and versatile synthetic biology toolkit, which expands the biocomputing capacity of Transcriptional Programming via the development of compressed and scalable multi-INPUT/OUTPUT logical operations.
more »
« less
Engineered systems of inducible anti-repressors for the next generation of biological programming
Abstract Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl β-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.
more »
« less
- PAR ID:
- 10190864
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Transcription rates are regulated by the interactions between RNA polymerase, sigma factor, and promoter DNA sequences in bacteria. However, it remains unclear how non-canonical sequence motifs collectively control transcription rates. Here, we combine massively parallel assays, biophysics, and machine learning to develop a 346-parameter model that predicts site-specific transcription initiation rates for any σ70promoter sequence, validated across 22132 bacterial promoters with diverse sequences. We apply the model to predict genetic context effects, design σ70promoters with desired transcription rates, and identify undesired promoters inside engineered genetic systems. The model provides a biophysical basis for understanding gene regulation in natural genetic systems and precise transcriptional control for engineering synthetic genetic systems.more » « less
-
Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budgetAbstract Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.more » « less
-
Abstract Bacteroidesspecies are prominent members of the human gut microbiota. The prevalence and stability ofBacteroidesin humans make them ideal candidates to engineer as programmable living therapeutics. Here we report a biotic decision-making technology in a community ofBacteroides(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 donorBacteroides, 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 inBacteroidesfor the development of bespoke therapeutic bacteria.more » « less
-
null (Ed.)Allosteric function is a critical component of many of the parts used to construct gene networks throughout synthetic biology. In this review, we discuss an emerging field of research and education, biomolecular systems engineering, that expands on the synthetic biology edifice—integrating workflows and strategies from protein engineering, chemical engineering, electrical engineering, and computer science principles. We focus on the role of engineered allosteric communication as it relates to transcriptional gene regulators—i.e., transcription factors and corresponding unit operations. In this review, we ( a) explore allosteric communication in the lactose repressor LacI topology, ( b) demonstrate how to leverage this understanding of allostery in the LacI system to engineer non-natural BUFFER and NOT logical operations, ( c) illustrate how engineering workflows can be used to confer alternate allosteric functions in disparate systems that share the LacI topology, and ( d) demonstrate how fundamental unit operations can be directed to form combinational logical operations.more » « less
