Biology subjects

Ajo-Franklin, C.

Publications and source records attributed to Ajo-Franklin, C..

8 recordsLinked to original sources

bioRxiv2025

Using domain insertion to create sulfite reductases that present chemical-dependent activities

Domain insertion can be used to create oxidoreductase switches whose charge transfer is dependent upon analyte binding. To date, most domain insertion studies have targeted relatively small proteins of known structure, so it remains unclear how to effectively use this protein engineering approach with large oligomeric oxidoreductases that require dynamic conformational changes for catalysis. To address this question, we studied the effect of domain insertion on the function of NADPH-dependent sulfite reductase (SiR) from Escherichia coli, a dodecameric protein containing eight hemoprotein subunits and four flavoprotein subunits. SiR mutational tolerance was first mapped using systematic peptide insertion, and a subset of variants retaining activity were subjected to domain insertion. When a ligand-binding domain was inserted at locations tolerant to peptide insertion, including sites proximal and distal from the intersubunit interfaces, more than half of the resulting variants presented cellular activity that is enhanced by an endocrine disruptor. This ligand-dependent synthesis of a redox-active metabolite could be monitored electrochemically from cells, illustrating how a single protein complex can be used to convert chemical information in the environment into a metabolite within cells that diffuses across the cell membrane and can be detected electrochemically.

synthetic biology↗

bioRxiv2024

Genetically modifying the protein matrix of macroscopic living materials to control their structure and rheological properties

The field of engineering living materials (ELMs) seeks to engineer cells to form macroscopic materials with tailorable structures and properties. While centimeter-scale ELMs can be grown from Caulobacter crescentus engineered to secrete a protein matrix, how the sequence of the protein matrix affects structural and rheological properties remains poorly understood. Here, we explore how changing the elastin-like polypeptide (ELP) length impacts ELM microstructure and viscoelastic behavior. We demonstrate that shortening ELP produces fibers almost 2x thicker than other variants, resulting in a stiffer material at rest. Interestingly, the mid-length ELP forms a complex structure with globules and multidirectional fibers with increased yield stress under flow conditions. Lengthening ELP creates thinner strands between cells with similar storage and loss moduli to the mid-length ELP. This study indicates that sequence-structure-property relationships in these ELMs are complex with few parallels to other biocomposite models. Furthermore, it highlights that fine-tuning genetic sequences can create significant differences in rheological properties, uncovering new design principles of ELMs.

synthetic biology↗

bioRxiv2023

Multichannel bioelectronic sensing using engineered Escherichia coli

By engineering extracellular electron transfer (EET) to be dependent on an analyte, researchers have developed whole cell bioelectronic sensors that sense hazards to human and environmental health1. However, these sensors regulate a single electron transfer pathway as an electrochemical channel, limiting the sensing information to a single analyte. To increase information content, we developed a multichannel bioelectronic sensor through which different chemicals regulate distinct extracellular electron transfer pathways within a single Escherichia coli cell. One channel utilizes the flavin synthesis pathway from Bacillus subtilis2 and the other a set of cytochromes constructing the Mtr pathway from Shewanella oneidensis3. We demonstrate an arsenite responsive promoter can control the Mtr pathway through activation of cytochrome CymA expression and a cadmium responsive promoter can control the flavin synthesis pathway4,5. The redox potential of flavin-mediated EET is different from that of CymA-mediated one6. This allowed for development of a redox-potential-dependent algorithm that distinguishes variable input signals of each analyte mediated by two EET pathways in vivo. This approach enables a 2-bit binary signal readout for real-time tracking throughout the entire sensing duration. Our multichannel bioelectronic sensor was able to accurately sense and distinguish different heavy metals in Brays Bayou water samples with a response time comparable to that in clean water. This multichannel bioelectronic sensors allow for simultaneous detection of different chemicals, significantly expanding information transmission and helping to safeguard human and environmental health.

bioengineering↗

bioRxiv2023

Selective bioelectronic sensing of quinone pharmaceuticals using extracellular electron transfer in Lactiplantibacillus plantarum

Redox-active small molecules containing quinone functional groups play important roles as pharmaceuticals, but can be toxic if overdosed. Despite the need for a fast and quantitative method to detect quinone and its derivatives, current sensing strategies are often slow and struggle to differentiate between structural analogs. Leveraging the discovery that microorganisms use certain quinones to perform extracellular electron transfer (EET), we investigated the use of Lactiplantibacillus plantarum as a whole-cell bioelectronic sensor to selectively sense quinone analogs. By tailoring the native EET pathway in L. plantarum, we enabled quantitative quinone sensing of 1,4-dihydroxy-2-naphthoic acid (DHNA) - a gut bifidogenic growth stimulator. We found that L. plantarum could respond to environmental DHNA within seconds, producing electronic signals that cover a 106 concentration range. This sensing capacity was robust in different assay media and allowed for continuous monitoring of DHNA concentrations. In a simulated gut environment containing a mixed pool of quinone derivatives, this tailored EET pathway can selectively sense pharmacologically relevant quinone analogs, such as DHNA and menadione, amongst other structurally similar quinone derivatives. We also developed a multivariate model to describe the mechanism behind this selectivity and found a predictable correlation between quinone physiochemical properties and the corresponding electronic signals. Our work presents a new strategy to selectively sense redox-active molecules using whole-cell bioelectronic sensors and opens the possibility of using probiotic L. plantarum for bioelectronic applications in human health. Significant StatementQuinone-containing pharmaceuticals show toxicity at high concentrations, making it important to quickly and accurately measure their concentration while distinguishing between analogs. To address this problem, we leveraged recent discoveries in electroactive bacteria to develop a novel concept for whole-cell sensing. This concept combines selectivity and specificity, enabling differentiation between analogs based on the temporal dynamic of electron transfer in living cells. With this strategy, we achieved selective detection of pharmacologically relevant quinones with distinct electronic signals for each analog. These signals were deciphered by a multivariate model to provide insight into the specific physiochemical properties of each analog. We envision that this new concept can be applied to other analytes for faster and more efficient sensing using electroactive whole cells.

bioengineering↗

bioRxiv2023

Lactiplantibacillus plantarum uses ecologically relevant, exogenous quinones for extracellular electron transfer

Extracellular electron transfer (EET) is a metabolic process that frequently uses quinones to couple intracellular redox reactions with extracellular electron acceptors. The physiological relevance of this metabolism for microorganisms that are capable of EET, but unable to synthesize their own quinones, remains to be determined. To address this question, we investigated quinone utilization by Lactiplantibacillus plantarum, a microorganism required for food fermentations, performs EET, and is also a quinone auxotroph. L. plantarum selectively used 1,4-dihydroxy-2-naphthoic acid (DHNA), 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), 1,4-naphthoquinone, and menadione for EET reduction of insoluble iron (ferrihydrite). However, those quinones used for EET also inhibited L. plantarum growth in non-aerated conditions. Transcriptomic analysis showed that DHNA induced oxidative stress in L. plantarum and this was alleviated by the inclusion of an electron acceptor, soluble ferric ammonium citrate (FeAC), in the laboratory culture medium. The presence of DHNA and FeAC during growth also induced L. plantarum EET metabolism, although activity was still dependent on the presence of exogenous electron shuttles. To determine whether quinone-producing bacteria frequently found together with L. plantarum in food fermentations could be a source of those electron shuttles, L. plantarum EET was measured after incubation with Lactococcus lactis and Leuconostoc mesenteroides. Quinone-producing L. lactis, but not a quinone-deficient L. lactis {Delta}menC mutant, increased L. plantarum ferrihydrite reduction and medium acidification through an EET-dependent mechanism. L. plantarum EET was also stimulated by L. mesenteroides, and this resulted in greater environmental acidification and transient increases in L. plantarum growth. Overall, our findings revealed that L. plantarum overcomes the toxic effects of exogenous quinones to use those compounds, including those made by related bacteria, for EET-conferred, ecological advantages during the early stages of food fermentations.

microbiology↗

bioRxiv2021

A de novo matrix for macroscopic living materials from bacteria

Summary ParagraphEngineered living materials (ELMs) are composites of living cells embedded in a biopolymer matrix that combine the desirable properties of natural biomaterials with non-natural, tailored properties. ELMs with a wide range of sophisticated biological functions have been created by engineering the embedded cells using synthetic biology. Engineering a de novo biomolecular matrix would offer control over material assembly, structure, and composition, thus enabling us to grow macroscopic ELMs with customizable mechanical properties. However, we have lacked the genetic tools and design rules to genetically encode a synthetic matrix that programs collective cell self-organization into macroscopic structures. Here we report growth of macroscopic ELMs from Caulobacter crescentus cells that display and secrete an engineered self-interacting protein. This protein formed an extracellular de novo matrix and assembled cells into hierarchically-ordered, centimeter-scale ELMs. We showed that the mechanical, catalytic, and morphological properties of these ELMs can be tuned through genetic modification of the matrix. Our work identifies novel genetic tools, design and assembly rules for growing macroscopic ELMs with both wide-ranging mechanical properties and customizable functions. We anticipate the modularity of this approach will permit the incorporation of different protein polymers in the de novo matrix, thus allowing to generate ELMs with a variety of desired structures and compositions of the bulk material. We envision specific matrix properties that can be combined synergistically with existing cellular functions to greatly expand the opportunities for ELMs in human health, energy, and the environment.

synthetic biology↗

bioRxiv2021

Real-time environmental monitoring of contaminants using living electronic sensors

Real-time chemical sensing is needed to counter the global threats posed by pollution. We combine synthetic biology and materials engineering to develop a living bioelectronic sensor platform with minute detection times. Escherichia coli was programmed to reduce an electrode in a chemical-dependent manner using a modular, eight-component, synthetic electron transport chain. This strain produced significantly more current upon exposure to thiosulfate, an anion that causes microbial blooms. Incorporating a protein switch into the synthetic pathway and encapsulation of microbes with electrodes and conductive nanomaterials yielded a living bioelectronic sensor that could detect an endocrine disruptor within two minutes in riverine water, implicating the signal as mass transfer limited. These findings provide a new platform for miniature, low-power sensors that safeguard ecological and human health. One Sentence SummaryChemicals are detected electrically using an allosterically-regulated electron transfer pathway in designer microbes.

synthetic biology↗

bioRxiv2020

Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

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 elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or 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 peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards the formation of cohesive engineered living materials. IMPORTANCEEngineered 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 towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

synthetic biology↗