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Results for “Synthetic Biology”

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bioRxiv2022

Engineered bacteria recruit and orchestrate anti-tumor immunity

Tumors employ multiple mechanisms to actively exclude or suppress adaptive immune cells involved in anti-tumor immunity. Strategies focused on overcoming these immunosuppressive or exclusion signals - through localized delivery of chemokines that directly recruit immune cells into the tumor microenvironment - remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor localized delivery, offering therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to produce and intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Intravenous or intratumoral delivery of bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) leads to the recruitment of activated T cells within tumors and offers therapeutic benefit in multiple mouse tumor models. Furthermore, we rationally target an additional step in the immune activation cascade - specifically, the presentation of tumor-derived antigens by dendritic cells - using a second engineered bacterial strain expressing CCL20. This combined targeting approach led to the recruitment of type 1 conventional dendritic cells and effectively synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to cooperatively recruit and activate both innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

cancer biology↗

bioRxiv2022

Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression

Super-enhancers (SEs) are a class of compound regulatory elements which control expression of key cell-identity genes. It remains unclear whether they are simply clusters of independent classical enhancers or whether SEs manifest emergent properties and should therefore be considered as a distinct class of element. Here, using synthetic biology and genome editing, we engineered the well characterised erythroid -globin SE at the endogenous -globin locus, removing all SE constituent elements in a mouse embryonic stem cell-line, to create a "blank canvas". This has allowed us to re-build the SE through individual and combinatorial reinsertion of its five elements (R1, R2, R3, Rm, R4), to test the importance of each constituents sequence and position within the locus. Each re-inserted element independently creates a region of open chromatin and binds its normal repertoire of transcription factors; however, we found a high degree of functional interdependence between the five constituents. Surprisingly, the two strongest -globin enhancers (R1 and R2) act sub-optimally both on their own and in combination, and although the other three elements (R3, Rm and R4) exhibit no discernible enhancer activity, they each exert a major positive effect in facilitating the activity of the classical enhancers (R1 and R2). This effect depends not simply on the sequence of each element but on their positions within the cluster. We propose that these "facilitators" are a novel form of regulatory element, important for ensuring the full activity of SEs, but distinct from conventional enhancer elements.

genetics↗

bioRxiv2022

A short-term decellularisation technique for porcine carotid arteries that conveys a structural stimulus to cells

In many cases, treatment for stenotic atherosclerotic lesions requires the use of bypass grafts to divert blood flow around the diseased vessel sections. Autologous vessels are considered the "gold standard" for bypass conduits; however, the shortage of healthy autologous vessels has resulted in an increasing focus on optimising synthetic, biological and/or tissue engineered vascular bypass grafts. While many of the previously published methods have been shown to fall short of producing an ideal TEVG, this report presents a decellularisation process that produces an acellular vascular graft that is efficient, cost effective, and could be readily automated. The resulting graft can be used "off the shelf", has preserved arterial structure and mechanical properties, and conforms to decellularisation criteria regarding the sufficient removal of cellular and genetic components. Additionally, the graft does not require any priming, supports molecular transport, can withstand supraphysiological pressures, and can support cell attachment and growth under physiological strain conditions whilst providing structural cues for cell adhesion and growth. Impact statementVascular disease remains the leading cause of mortality worldwide. In the absence of suitable autologous vessels, there currently exists a clear clinical need for off the shelf vascular grafts that can successfully bypass diseased arteries. This paper outlines a short-term method for obtaining such a graft. The technique used involves decellularising porcine carotid arteries whilst preserving arterial structure and mechanical properties and is compliant with the international standard for implantable vascular prostheses: EN ISO 7198:2017. Additionally, this protocol is cost and time effective, and produces reproducible "ready to use" acellular grafts that support molecular transport, can withstand supraphysiological pressures, and can support cell attachment and growth with controlled structural cues under physiological strain conditions.

bioengineering↗

bioRxiv2022

Studying Pathogens Degrades BLAST-based Pathogen Identification

As synthetic biology becomes increasingly capable and accessible, it is likewise increasingly critical to be able to make accurate biosecurity determinations regarding the pathogenicity or toxicity of particular nucleic acid or amino acid sequences. At present, this is typically done using the BLAST algorithm to determine the best match with sequences in the NCBI databases. Neither BLAST nor the NCBI databases, however, are actually designed for biosafety determination. Critically, taxonomic errors or ambiguities in the NCBI databases can also cause errors in BLAST-based taxonomic categorization. With heavily studied taxa and frequently used biotechnology tools, even low frequency taxonomic categorization issues can lead to high rates of errors in biosecurity decision-making. Here we focus on the implications for false positives, finding that NCBI BLAST will now incorrectly categorize a number of commonly used biotechnology tool sequences as the pathogens or toxins with which they have been used. Paradoxically, this implies that problems are expected to be most acute for the pathogens and toxins of highest interest and the most widely used biotechnology tools. We thus conclude that biosecurity tools should shift away from BLAST against NCBI and towards new methods that are specifically tailored for biosafety purposes.

bioinformatics↗

bioRxiv2022

The Spatial Organization of Bacterial Transcriptional Regulatory Networks

Transcriptional regulatory network (TRN) is the central pivot of a prokaryotic organism to receive, process and respond to internal and external environmental information. However, little is known about its spatial organization so far. In recent years, chromatin interaction data of bacteria such as Escherichia coli and Bacillus subtilis have been published, making it possible to study the spatial organization of bacterial transcriptional regulatory networks. By combining TRNs and chromatin interaction data of E. coli and B. subtilis, we explored the spatial organization characteristics of bacterial TRNs in many aspects such as regulation directions (positive and negative), central nodes (hubs, bottlenecks), hierarchical levels (top, middle, bottom) and network motifs (feed-forward loops and single input modules) of the TRNs and found that the bacterial TRNs have a variety of stable spatial organization features under different physiological conditions which may be closely related with basic life activities. Our findings provided new insights into the connection between transcriptional regulation and the spatial organization of chromosome in bacteria, and might serve as a foundation for spatial-distance-based gene circuit design in synthetic biology.

systems biology↗

bioRxiv2022

Light-dependent Control of Bacterial Expression at the mRNA Level

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several ten-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that generally control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

biochemistry↗

bioRxiv2022

Neural Network-Derived Potts Models for Structure-Based Protein Design using Backbone Atomic Coordinates and Tertiary Motifs

AO_SCPLOWBSTRACTC_SCPLOWDesigning novel proteins to perform desired functions, such as binding or catalysis, is a major goal in synthetic biology. A variety of computational approaches can aid in this task. An energy-based framework rooted in the sequence-structure statistics of tertiary motifs (TERMs) can be used for sequence design on pre-defined backbones. Neural network models that use backbone coordinate-derived features provide another way to design new proteins. In this work, we combine the two methods to make neural structure-based models more suitable for protein design. Specifically, we supplement backbone-coordinate features with TERM-derived data, as inputs, and we generate energy functions as outputs. We present two architectures that generate Potts models over the sequence space: TERMinator, which uses both TERM-based and coordinate-based information, and COORDinator, which uses only coordinate-based information. Using these two models, we demonstrate that TERMs can be utilized to improve native sequence recovery performance of neural models. Furthermore, we demonstrate that sequences designed by TERMinator are predicted to fold to their target structures by AlphaFold. Finally, we show that both TERMinator and COORDinator learn notions of energetics, and these methods can be fine-tuned on experimental data to improve predictions. Our results suggest that using TERM-based and coordinate-based features together may be beneficial for protein design and that structure-based neural models that produce Potts energy tables have utility for flexible applications in protein science. CodeCode will be made publically available at https://github.com/alexjli/terminator_public

biophysics↗

bioRxiv2022

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

RNA nanotechnology uses motifs from nature as well as aptamers from in vitro selection to construct nanostructures and devices for applications in RNA medicine and synthetic biology. The RNA origami method allows cotranscriptional folding of large RNA scaffolds that can position functional motifs in a precise manner, which has been verified by Forster Resonance Energy Transfer (FRET) between fluorescent aptamers. Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA nanostructures. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight. Here, we place aptamers on the RNA origami scaffolds to increase the contrast for cryo-EM and solve the structure of a new Broccoli-Pepper FRET pair. We identify different modes of ligand binding of the two aptamers and verify by selective probing. 3D variability analysis of the cryo-EM data show that the relative position between the two bound fluorophores on the origami fluctuate by only 3.5 Angstrom. Our results demonstrate the use of RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision. The Broccoli-Pepper apta-FRET pair has potential use for developing advanced sensors that are sensitive to small conformational changes.

biochemistry↗

bioRxiv2022

A dueling-competent signal-sensing module guides precise delivery of cargo proteins into target cells by engineered Pseudomonas aeruginosa

To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here, we introduce a highly-selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells in a mixed population. These data demonstrate that the DUEC cell is not only a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

microbiology↗

bioRxiv2022

Environment-dependent epistasis increases phenotypic diversity in gene regulatory networks

Mutations to gene regulatory networks can be maladaptive or a source of evolutionary novelty. Epistasis confounds our understanding of how mutations impact the expression patterns of gene regulatory networks, because such nonlinearities make it difficult to predict the combined phenotypic effects of mutations based on knowledge of the mutations individual effects. This challenge is exacerbated by the dependence of epistasis on the environment, which is particularly germane to gene regulatory networks that interpret signals in space or time. To help fill this knowledge gap, we used the toolkit of synthetic biology to systematically assay the effects of pairwise and triplet combinations of mutant genotypes on the expression pattern of a gene regulatory network expressed in Escherichia coli that interprets an inducer gradient across a spatial domain. We uncovered a preponderance of epistasis in both pairwise and triplet combinations that can switch in magnitude and sign across the inducer gradient to produce a greater diversity of expression pattern phenotypes than would be possible in the absence of such environment-dependent epistasis. We discuss our findings in the context of the evolution of hybrid incompatibilities and evolutionary novelties, arguing that environment-dependent epistasis is likely an important cause of both phenomena in gene regulatory networks.

evolutionary biology↗

bioRxiv2022

Fake it to break it: mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the non-structural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic-biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titres and transmission potential in mosquitoes.

microbiology↗

bioRxiv2022

Engineering a plant polyketide synthase for the biosynthesis of methylated flavonoids

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in Escherichia coli from fed O-methylated cinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare, in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in E. coli towards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids.

biochemistry↗

bioRxiv2022

Osmotic pressure enables high yield assembly of giant vesicles in solutions of physiological ionic strengths

Giant unilamellar vesicles (GUVs) are micrometer scale minimal cellular mimics that are useful for synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100-150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or in the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six assisting polymeric and small molecule compounds on the molar yields of GUVs composed of three different lipid mixtures using high resolution confocal microscopy and large dataset image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 degrees Celsius, whereas the small molecule compound was ineffective. Low gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10 %. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs support our models prediction. In addition, polymer specific interactions with the substrate and the lipid mixture effects yields. The uncovered mechanistic insight provides a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.

biophysics↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2022

Split selectable marker mediated gene stacking in plants

Plant synthetic biology and genetic engineering depend on the controlled expression of transgenes of interest, which relies heavily on the use of Agrobacterium-mediated transformation. Here we establish a novel split selectable marker system using protein splicing elements called "inteins" for Agrobacterium-mediated co-transformation in plants. This method enables robust co-transformation in Arabidopsis and poplar, providing a novel strategy for the simultaneous insertion of multiple genes into both herbaceous and woody plants.

plant biology↗

bioRxiv2022

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The [~]100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-located abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

microbiology↗

bioRxiv2022

Illuminating protein space with a programmable generative model

Three billion years of evolution have produced a tremendous diversity of protein molecules, and yet the full potential of this molecular class is likely far greater. Accessing this potential has been challenging for computation and experiments because the space of possible protein molecules is much larger than the space of those likely to host function. Here we introduce Chroma, a generative model for proteins and protein complexes that can directly sample novel protein structures and sequences and that can be conditioned to steer the generative process towards desired properties and functions. To enable this, we introduce a diffusion process that respects the conformational statistics of polymer ensembles, an efficient neural architecture for molecular systems based on random graph neural networks that enables long-range reasoning with sub-quadratic scaling, equivariant layers for efficiently synthesizing 3D structures of proteins from predicted inter-residue geometries, and a general low-temperature sampling algorithm for diffusion models. We suggest that Chroma can effectively realize protein design as Bayesian inference under external constraints, which can involve symmetries, substructure, shape, semantics, and even natural language prompts. With this unified approach, we hope to accelerate the prospect of programming protein matter for human health, materials science, and synthetic biology.

bioinformatics↗

bioRxiv2023

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Flavin adenine dinucleotide (FAD), an essential cofactor in cellular metabolism, catalyses a wide range of redox reactions. The organic synthesis of FAD is typically conducted by coupling flavin mononucleotide (FMN) and adenosine monophosphate. The reported synthesis routes have certain limitations such as multiple reaction steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues using chemical and enzymatic methods with readily available starting materials achieved in 1-3 steps with moderate yields (10-51%). Further, we demonstrate that Escherichia coli glutathione reductase can use these analogues to catalyse the reduction of glutathione. Finally, we show that FAD nucleobase analogues can also be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology applications.

molecular biology↗