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1. Next generation synthetic memory via intercepting recombinase function

   Short, A.E. (October 2023, Nature communications)

   Ross Cloney (Ed.)

   Here we present a technology to facilitate synthetic memory in a living system via repurposing Transcriptional Programming (i.e., our decision-making technology) parts, to regulate (intercept) recombinase function post-translation. We show that interception synthetic memory can facilitate programmable loss-of-function via site-specific deletion, programmable gain-of-function by way of site-specific inversion, and synthetic memory operations with nested Boolean logical operations. We can expand interception synthetic memory capacity more than 5-fold for a single recombinase, with reconfiguration specificity for multiple sites in parallel. Interception synthetic memory is ~10-times faster than previous generations of recombinase-based memory. We posit that the faster recombination speed of our next-generation memory technology is due to the post-translational regulation of recombinase function. This iteration of synthetic memory is complementary to decision-making via Transcriptional Programming – thus can be used to develop intelligent synthetic biological systems for myriad applications. 

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2. Engineering intelligent chassis cells via recombinase-based MEMORY circuits

   https://doi.org/10.1038/s41467-024-46755-1  

   Huang, Brian D.; Kim, Dowan; Yu, Yongjoon; Wilson, Corey J. (March 2024, Nature Communications)

   Abstract Synthetic biologists seek to engineer intelligent living systems capable of decision-making, communication, and memory. Separate technologies exist for each tenet of intelligence; however, the unification of all three properties in a living system has not been achieved. Here, we engineer completely intelligentEscherichia colistrains that harbor six orthogonal and inducible genome-integrated recombinases, forming Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY). MEMORY chassis cells facilitate intelligence via the discrete multi-input regulation of recombinase functions enabling inheritable DNA inversions, deletions, and genomic insertions. MEMORY cells can achieve programmable and permanent gain (or loss) of functions extrachromosomally or from a specific genomic locus, without the loss or modification of the MEMORY platform – enabling the sequential programming and reprogramming of DNA circuits within the cell. We demonstrate all three tenets of intelligence via a probiotic (Nissle 1917) MEMORY strain capable of information exchange with the gastrointestinal commensalBacteroides thetaiotaomicron. 

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3. Quantum-inspired logic for advanced Transcriptional Programming

   https://doi.org/10.1093/nar/gkaf440  

   Milner, Prasaad_T; Kim, Dowan; Wilson, Corey J. (May 2025, Nucleic Acids Research)

   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. 

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4. Transcriptional programming in a Bacteroides consortium

   https://doi.org/10.1038/s41467-022-31614-8  

   Huang, Brian D.; Groseclose, Thomas M.; Wilson, Corey J. (July 2022, Nature Communications)

   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. 

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5. DASH : a versatile and high‐capacity gene stacking system for plant synthetic biology

   https://doi.org/10.1111/pbi.70179  

   Zhao, Chengsong; Stepanova, Anna_N; Alonso, Jose_M (June 2025, Plant Biotechnology Journal)

   Summary DNA assembly systems based on the Golden Gate method are popular in synthetic biology but have several limitations: small insert size, incompatibility with other cloning platforms, DNA domestication requirement, generation of fusion scars, and lack of post‐assembly modification. To address these obstacles, we present the DASH assembly toolset, which combines features of Golden Gate‐based cloning, recombineering, and site‐specific recombinase systems. We developed (1) a set of donor vectors based on the GoldenBraid platform, (2) an acceptor vector derived from the plant transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, and (3) a re‐engineered recombineering‐readyE. colistrain, CZ105, based on SW105. The initial assembly steps are carried out using the donor vectors following standard GoldenBraid assembly procedures. Importantly, existing parts and transcriptional units created using compatible Golden Gate‐based systems can be transferred to the DASH donor vectors using standard single‐tube restriction/ligation reactions. The cargo DNA from a DASH donor vector is then efficiently transferredin vivoinE. coliinto the acceptor vector by the sequential action of a rhamnose‐inducible phage‐derived PhiC31 integrase and arabinose‐inducible yeast‐derived Flippase (FLP) recombinase using CZ105. Furthermore, recombineering‐based post‐assembly modification, including the removal of undesirable scars, is greatly simplified. To demonstrate the utility of the DASH system, a 116 kilobase (kb) DNA construct harbouring a 97 kb cargo consisting of 35 transcriptional units was generated. One of the coding DNA sequences (CDSs) in the final assembly was replaced through recombineering, and thein plantafunctionality of the entire construct was tested in both transient and stable transformants. 

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