Journal: ACS synthetic biology
Sustainable and personally tailored materials production is an emerging challenge to society. Living organisms can produce and pattern an extraordinarily wide range of different molecules in a sustainable way. These natural systems offer an abundant source of inspiration for the development of new environmentally-friendly materials production techniques. In this paper, we describe the first steps towards the 3-dimensional printing of bacterial cultures for materials production and patterning. This methodology combines the capability of bacteria to form new materials with the reproducibility and tailored approach of 3D printing systems. For this purpose, a commercial 3D printer was modified for bacterial systems, and new alginate-based bio-ink chemistry was developed. Printing temperature, printhead speed, and bio-ink extrusion rate were all adapted and customized to maximize bacterial health and spatial resolution of printed structures. Our combination of 3D printing technology with biological systems enables a sustainable approach for the production of numerous new materials.
The volumetric heating values of today’s biofuels are too low to power energy-intensive aircraft, rockets, and missiles. Recently, pinene dimers were shown to have a volumetric heating value similar to that of the tactical fuel JP-10. To provide a sustainable source of pinene, we engineered Escherichia coli for pinene production. We combinatorially expressed three pinene synthases (PS) and three geranyl diphosphate synthases (GPPS), with the best combination achieving ∼28 mg/L of pinene. We speculated that pinene toxicity was limiting production; however, toxicity should not be limiting at current titers. Because GPPS is inhibited by geranyl diphosphate (GPP) and to increase flux through the pathway, we combinatorially constructed GPPS-PS protein fusions. The Abies grandis GPPS-PS fusion produced 32 mg/L of pinene, a 6-fold improvement over the highest titer previously reported in engineered E. coli. Finally, we investigated the pinene isomer ratio of our pinene-producing microbe and discovered that the isomer profile is determined not only by the identity of the PS used but also by the identity of the GPPS with which the PS is paired. We demonstrated that the GPP concentration available to PS for cyclization alters the pinene isomer ratio.
Rose has been entwined with human culture and history. “Blue rose” in English signifies unattainable hope or impossible mission as it does not exist naturally and is not breedable regardless of centuries of efforts by gardeners. With the knowledge of genes and enzymes involved in flower pigmentation and modern genetic technologies, synthetic biologists have undertaken the challenge of producing blue rose by engineering the complicated vacuolar flavonoid pigmentation pathway and resulted in a mauve coloured rose. A completely different strategy presented in this study employs a dual expression plasmid containing bacterial idgS and sfp genes. The holo-IdgS, activated by Sfp from its apo-form, is a functional non-ribosomal peptide synthetase that converts L-glutamine into a blue pigment indigoidine. Expression of these genes upon petal injection with agro-infiltration solution generates blue-hued rose flowers. We envision that implementing this proof-of-concept with obligatory modifications may have tremendous impact in floriculture to achieve historic milestone in rose breeding.
The ability to regulate endogenous gene expression is critical in biological research. Existing technologies, such as RNA interference, zinc-finger regulators, transcription-activator-like effectors, and CRISPR-mediated regulation, though proved to be competent in significantly altering expression levels, do not provide a quantitative adjustment of regulation effect. As a solution to this problem, we place CRISPR-mediated interference under the control of blue light: while dCas9 protein is constitutively expressed, guide RNA transcription is regulated by YF1-FixJ-PFixK2, a blue light responding system. With a computer-controlled luminous device, the quantitative relationship between target gene expression and light intensity has been determined. As the light intensifies, the expression level of target gene gradually ascends. This remarkable property enables sensor-CRISPRi to accurately interrogate cellular activities.
Recently, synthetic biologists have developed the Synthetic Biology Open Language (SBOL), a data exchange standard for descriptions of genetic parts, devices, modules, and systems. The goals of this standard are to allow scientists to exchange designs of biological parts and systems, to facilitate the storage of genetic designs in repositories, and to facilitate the description of genetic designs in publications. In order to achieve these goals, the development of an infrastructure to store, retrieve, and exchange SBOL data is necessary. To address this problem, we have developed the SBOL Stack, a Resource Description Framework (RDF) database specifically designed for the storage, integration, and publication of SBOL data. This database allows users to define a library of synthetic parts and designs as a service, to share SBOL data with collaborators, and to store designs of biological systems locally. The database also allows external data sources to be integrated by mapping them to the SBOL data model. The SBOL Stack includes two Web interfaces: the SBOL Stack API and SynBioHub. While the former is designed for developers, the latter allows users to upload new SBOL biological designs, download SBOL documents, search by keyword, and visualize SBOL data. Since the SBOL Stack is based on semantic Web technology, the inherent distributed querying functionality of RDF databases can be used to allow different SBOL stack databases to be queried simultaneously, and therefore, data can be shared between different institutes, centers, or other users.
Engineered T cells are transforming broad fields in biomedicine, yet our ability to control cellular activity at specific anatomical sites remains limited. Here we engineer thermal gene switches to allow spatial and remote control of transcriptional activity using pulses of heat. These gene switches are constructed from the heat shock protein HSP70B' (HSPA6) promoter, show negligible basal transcriptional activity, and activate within an elevated temperature window of 40-45°C. Using engineered Jurkat T cells implanted in vivo, we use plasmonic photothermal heating to trigger gene expression at specific sites to levels greater than 200-fold. We show that delivery of heat as thermal pulse trains significantly increase cellular thermal tolerance compared to continuous heating curves with identical area-under-the-curve (AUC), enabling long-term control of gene expression in Jurkat T cells. This approach expands the toolkit of remotely controlled genetic devices for basic and translational applications in synthetic immunology.
Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees ( Apis mellifera) and bumble bees ( Bombus sp.). Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella apis, and Serratia strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated knockout of an S. alvi adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities.
Novel approaches are needed to combat antibiotic resistance. Here, we describe a computational-experimental framework for the discovery of novel cryptic antimicrobial peptides (AMPs). The computational platform, based on previously validated antimicrobial scoring functions, indicated the activation peptide of pepsin A, the main human stomach protease, and its N- and C-terminal halves as antimicrobial peptides. The three peptides from pepsinogen A3 isoform were prepared in a recombinant form using a fusion carrier specifically developed to express toxic peptides in Escherichia coli. Recombinant pepsinogen A3-derived peptides proved to be wide-spectrum antimicrobial agents with MIC values in the range 1.56-50 μM (1.56-12.5 μM for the whole activation peptide). Moreover, the activation peptide was bactericidal at pH 3.5 for relevant foodborne pathogens, suggesting that this new class of previously unexplored AMPs may contribute to microbial surveillance within the human stomach. The peptides showed no toxicity toward human cells and exhibited anti-infective activity in vivo, reducing by up to 4 orders of magnitude the bacterial load in a mouse skin infection model. These peptides thus represent a promising new class of antibiotics. We envision that computationally guided data mining approaches such as the one described here will lead to the discovery of antibiotics from previously unexplored sources.
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron-sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae where transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step towards obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.