BioSciences Publications

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https://hdl.handle.net/1911/78269

BioSciences faculty publications. For works published before Summer 2014, please see the Biochemistry & Cell Biology and Ecology & Evolutionary Biology collections.

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A de novo matrix for macroscopic living materials from bacteria
(Springer Nature, 2022) Molinari, Sara; Tesoriero, Robert F.; Li, Dong; Sridhar, Swetha; Cai, Rong; Soman, Jayashree; Ryan, Kathleen R.; Ashby, Paul D.; Ajo-Franklin, Caroline M.
Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.
A Framework for the Systematic Selection of Biosensor Chassis for Environmental Synthetic Biology
(American Chemical Society, 2022) Sridhar, Swetha; Ajo-Franklin, Caroline M.; Masiello, Caroline A.
Microbial biosensors sense and report exposures to stimuli, thereby facilitating our understanding of environmental processes. Successful design and deployment of biosensors hinge on the persistence of the microbial host of the genetic circuit, termed the chassis. However, model chassis organisms may persist poorly in environmental conditions. In contrast, non-model organisms persist better in environmental conditions but are limited by other challenges, such as genetic intractability and part unavailability. Here we identify ecological, metabolic, and genetic constraints for chassis development and propose a conceptual framework for the systematic selection of environmental biosensor chassis. We identify key challenges with using current model chassis and delineate major points of conflict in choosing the most suitable organisms as chassis for environmental biosensing. This framework provides a way forward in the selection of biosensor chassis for environmental synthetic biology.
A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step
(Elsevier, 2020) Su, Lin; Fukushima, Tatsuya; Ajo-Franklin, Caroline M.
Bioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell−1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.
Controlled and Stable Patterning of Diverse Inorganic Nanocrystals on Crystalline Two-Dimensional Protein Arrays
(American Chemical Society, 2021) Mann, Victor R.; Manea, Francesca; Borys, Nicholas J.; Ajo-Franklin, Caroline M.; Cohen, Bruce E.
Controlled patterning of nanoparticles on bioassemblies enables synthesis of complex materials for applications in optics, nanoelectronics, and sensing. Biomolecular self-assembly offers molecular control for engineering patterned nanomaterials, but current approaches have been limited in their ability to combine high nanoparticle coverage with generality that enables incorporation of multiple nanoparticle types. Here, we synthesize photonic materials on crystalline two-dimensional (2D) protein sheets using orthogonal bioconjugation reactions, organizing quantum dots (QDs), gold nanoparticles (AuNPs), and upconverting nanoparticles along the surface-layer (S-layer) protein SbsB from the extremophile Geobacillus stearothermophilus. We use electron and optical microscopy to show that isopeptide bond-forming SpyCatcher and SnoopCatcher systems enable the simultaneous and controlled conjugation of multiple types of nanoparticles (NPs) at high densities along the SbsB sheets. These NP conjugation reactions are orthogonal to each other and to Au–thiol bond formation, allowing tailorable nanoparticle combinations at sufficient labeling efficiencies to permit optical interactions between nanoparticles. Fluorescence lifetime imaging of SbsB sheets conjugated to QDs and AuNPs at distinct attachment sites shows spatially heterogeneous QD emission, with shorter radiative decays and brighter fluorescence arising from plasmonic enhancement at short interparticle distances. This specific, stable, and efficient conjugation of NPs to 2D protein sheets enables the exploration of interactions between pairs of nanoparticles at defined distances for the engineering of protein-based photonic nanomaterials.
Electronic control of redox reactions inside Escherichia coli using a genetic module
(Public Library of Science, 2021) Baruch, Moshe; Tejedor-Sanz, Sara; Su, Lin; Ajo-Franklin, Caroline M.; Institute for Biosciences and Bioengineering
Microorganisms regulate the redox state of different biomolecules to precisely control biological processes. These processes can be modulated by electrochemically coupling intracellular biomolecules to an external electrode, but current approaches afford only limited control and specificity. Here we describe specific electrochemical control of the reduction of intracellular biomolecules in Escherichia coli through introduction of a heterologous electron transfer pathway. E. coli expressing cymAmtrCAB from Shewanella oneidensis MR-1 consumed electrons directly from a cathode when fumarate or nitrate, both intracellular electron acceptors, were present. The fumarate-triggered current consumption occurred only when fumarate reductase was present, indicating all the electrons passed through this enzyme. Moreover, CymAMtrCAB-expressing E. coli used current to stoichiometrically reduce nitrate. Thus, our work introduces a modular genetic tool to reduce a specific intracellular redox molecule with an electrode, opening the possibility of electronically controlling biological processes such as biosynthesis and growth in any microorganism.
Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
(American Society for Microbiology, 2021) Orozco-Hidalgo, Maria Teresa; Charrier, Marimikel; Tjahjono, Nicholas; Tesoriero, Robert F.; Li, Dong; Molinari, Sara; Ryan, Kathleen R.; Ashby, Paul D.; Rad, Behzad; Ajo-Franklin, Caroline M.
The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(−)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.
Solution-Deposited and Patternable Conductive Polymer Thin-Film Electrodes for Microbial Bioelectronics
(Wiley, 2022) Tseng, Chia-Ping; Liu, Fangxin; Zhang, Xu; Huang, Po-Chun; Campbell, Ian; Li, Yilin; Atkinson, Joshua T.; Terlier, Tanguy; Ajo-Franklin, Caroline M.; Silberg, Jonathan J.; Verduzco, Rafael
Microbial bioelectronic devices integrate naturally occurring or synthetically engineered electroactive microbes with microelectronics. These devices have a broad range of potential applications, but engineering the biotic–abiotic interface for biocompatibility, adhesion, electron transfer, and maximum surface area remains a challenge. Prior approaches to interface modification lack simple processability, the ability to pattern the materials, and/or a significant enhancement in currents. Here, a novel conductive polymer coating that significantly enhances current densities relative to unmodified electrodes in microbial bioelectronics is reported. The coating is based on a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) crosslinked with poly(2-hydroxyethylacrylate) (PHEA) along with a thin polydopamine (PDA) layer for adhesion to an underlying indium tin oxide (ITO) electrode. When used as an interface layer with the current-producing bacterium Shewanella oneidensis MR-1, this material produces a 178-fold increase in the current density compared to unmodified electrodes, a current gain that is higher than previously reported thin-film 2D coatings and 3D conductive polymer coatings. The chemistry, morphology, and electronic properties of the coatings are characterized and the implementation of these coated electrodes for use in microbial fuel cells, multiplexed bioelectronic devices, and organic electrochemical transistor based microbial sensors are demonstrated. It is envisioned that this simple coating will advance the development of microbial bioelectronic devices.

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Browsing BioSciences Publications by Author "Ajo-Franklin, Caroline M."

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