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Results for “Synthetic Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 289 records · Page 16Linked to original sources

bioRxiv2016

Control of Lipid Domain Organization by a Biomimetic Contractile Actomyosin Cortex

The cell membrane is a heterogeneously organized composite with lipid-protein micro-domains. The contractile actin cortex may govern the lateral organization of these domains in the cell membrane, yet the underlying mechanisms are not known. We recently reconstituted minimal actin cortices (MACs) (Vogel et al, 2013b) and here advanced our assay to investigate effects of rearranging actin filaments on the lateral membrane organization by introducing various phase-separated lipid mono-and bilayers to the MACs. The addition of actin filaments reorganized membrane domains. We found that the process reached a steady state where line tension and lateral crowding balanced. Moreover, the phase boundary allowed myosin driven actin filament rearrangements to actively move individual lipid domains, often accompanied by their shape change, fusion or splitting. Our findings illustrate how actin cortex remodeling in cells may control dynamic rearrangements of lipids and other molecules inside domains without directly binding to actin filaments.

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bioRxiv2016

A highly parallel strategy for storage of digital information in living cells

Encoding arbitrary digital information in DNA has attracted attention as a potential avenue for large scale and long term data storage. However, in order to enable DNA data storage technologies there needs to be improvements in data storage fidelity (tolerance to mutation), the facility of writing and reading the data (biases and systematic error arising from synthesis and sequencing), and overall scalability. To this end, we have developed and implemented an encoding scheme that is suitable for detecting and correcting errors that may arise during storage, writing, and reading, such as those arising from nucleotide substitutions, insertions, and deletions. We propose a scheme for parallelized long term storage of encoded sequences that relies on overlaps rather than the address blocks found in previously published work. Using computer simulations, we illustrate the encoding, sequencing, decoding, and recovery of encoded information, ultimately demonstrating the possibility of a successful round-trip read/write. These demonstrations show that in theory a precise control over error tolerance is possible. Even after simulated degradation of DNA, recovery of original data is possible owing to the error correction capabilities built into the encoding strategy. A secondary advantage of our method is that the statistical characteristics (such as repetitiveness and GC-composition) of encoded sequences can also be tailored without sacrificing the overall ability to store large amounts of data. Finally, the combination of the overlap-based partitioning of data with the LZMA compression that is integral to encoding means that the entire sequence must be present for successful decoding. This feature enables inordinately strong encryptions. As a potential application, an encrypted pathogen genome could be could be distributed and carried by cells without danger of being expressed, and could not even be read out in the absence of the entire DNA consortium.

synthetic biology↗

bioRxiv2017

Tuning of recombinant protein expression in Escherichia coli by manipulating transcription, translation initiation rates and incorporation of non-canonical amino acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. Based on the results, we developed a biophysical model, which suggests that the density of co-transcriptional translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

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bioRxiv2017

Daisyfield gene drive systems harness repeated genomic elements as a generational clock to limit spread

Methods of altering wild populations are most useful when inherently limited to local geographic areas. Here we describe a novel form of gene drive based on the introduction of multiple copies of an engineered daisy sequence into repeated elements of the genome. Each introduced copy encodes guide RNAs that target one or more engineered loci carrying the CRISPR nuclease gene and the desired traits. When organisms encoding a drive system are released into the environment, each generation of mating with wild-type organisms will reduce the average number of the guide RNA elements per daisyfield organism by half, serving as a generational clock. The loci encoding the nuclease and payload will exhibit drive only as long as a single copy remains, placing an inherent limit on the extent of spread.

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bioRxiv2017

Engineered shell proteins confer improved encapsulated pathway behavior in a bacterial microcompartment

Bacterial microcompartments are a class of proteinaceous organelles comprising a characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous pathways, but to do so, it is critical to understand how to control diffusion in and out of the microcompartment organelle. To this end, we explored how small differences in the shell protein structure result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2- propanediol, likely by controlling diffusion of one or more metabolites. This study highlights the success of two strategies to engineer microcompartment control over metabolite transport: altering the existing shell protein pore via mutation of the pore-lining residues, and generating chimeras using shell proteins with the desired pores.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC=\"FIGDIR/small/106716_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@1f69a12org.highwire.dtl.DTLVardef@fd5560org.highwire.dtl.DTLVardef@1f01d6aorg.highwire.dtl.DTLVardef@11b442c_HPS_FORMAT_FIGEXP M_FIG TOC Abstract Graphic C_FIG

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bioRxiv2017

Investigating circadian rhythmicity in pain sensitivity using a neural circuit model for spinal cord processing of pain

Primary processing of painful stimulation occurs in the dorsal horn of the spinal cord. In this article, we introduce mathematical models of the neural circuitry in the dorsal horn responsible for processing nerve fiber inputs from noxious stimulation of peripheral tissues and generating the resultant pain signal. The differential equation models describe the average firing rates of excitatory and inhibitory interneuron populations, as well as the wide dynamic range (WDR) neurons whose output correlates with the pain signal. The temporal profile of inputs on the different afferent nerve fibers that signal noxious and innocuous stimulation and the excitability properties of the included neuronal populations are constrained by experimental results. We consider models for the spinal cord circuit in isolation and when top-down inputs from higher brain areas that modulate pain processing are included. We validate the models by replicating experimentally observed phenomena of A fiber inhibition of pain and wind-up. We then use the models to investigate mechanisms for the observed phase shift in circadian rhythmicity of pain that occurs with neuropathic pain conditions. Our results suggest that changes in neuropathic pain rhythmicity can occur through dysregulation of inhibition within the dorsal horn circuit.

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bioRxiv2017

Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

Serine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.

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bioRxiv2017

A Spatially Localized Architecture for Fast and Modular Computation at the Molecular Scale

Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signaling networks. Here we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations, and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbors, identical DNA hairpins can be reused across circuits. Colocalization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate that our approach will motivate the use of spatial constraints in molecular engineering more broadly, bringing embedded molecular control circuits closer to applications.

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bioRxiv2017

CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

Interleukin 18 (IL18) is known to induce the expression of interferon-{gamma} (IFNG), but its effects on T cell proliferation and costimulation are not completely understood. In this study, we demonstrate that ectopic expression of IL18 in CART cells caused significant T cell proliferation in vitro and in vivo, and enhanced antitumor effects in xenograft models. Moreover, IL18 mediated T cell expansion required neither tumor antigen nor CAR expression, and produced severe GVHD in NSG mice. Furthermore, recombinant IL18 costimulated IFNG secretion and proliferation of anti-CD3 beads treated T cells. Interestingly, IL18 costimulation could expand purified CD4 T cells, but not CD8 T cells. However, CD8 T cells proliferated greater than CD4 T cells in magnitude within bulk T cells, suggesting CD4 help effect was involved. Using CRISPR/Cas9 gene editing, we confirmed that IL18-driven expansion was both TCR and IL18 receptor (IL18R) dependent. Importantly, we demonstrated that TCR-deficient, IL18-expressing CD19 CART cells exhibited remarkable proliferation and persistent antitumor activity against CD19-expressing tumor cells in vivo, without eliciting any detectable GVHD symptom. Finally, we describe APACHE T cells, a novel strategy for coupling IL18 expression in CART cells to antigen stimulation, thereby limiting potential toxicity associated with persistent IL18 production. In sum, our study supports human IL18 as a T cell costimulatory cytokine for fueling CART therapy.

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bioRxiv2017

Engineering pulsatile communication in bacterial consortia

Lux-type quorum sensing systems enable communication in bacteria with only two protein components: a signal synthase and an inducible transcription activator. The simplicity of these systems makes them a popular choice for engineering collaborative behaviors in synthetic bacterial consortia, such as photographic edge detection and synchronized oscillation. To add to this body of work, we propose a pulsatile communication circuit that enables dynamic patterning and long-distance communication analogous to action potentials traveling through nerve tissue. We employed a model-driven design paradigm involving incremental characterization of in vivo design candidates with increasing circuit complexity. Beginning with a simple inducible reporter system, we screened a small number of circuits varying in their promoter and ribosomal binding site (RBS) strengths. From this candidate pool, we selected a candidate to be the seed network for the subsequent round of more complex circuit variants, likewise variable in promoter and RBS strengths. The selection criteria at each level of complexity is tailored to optimize a different desirable performance characteristic. By this approach we individually optimized reporter signal-to-background ratio, pulsatile response to induction, and quiescent basal transcription, avoiding large library screens while ensuring robust performance of the composite circuit.

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bioRxiv2017

Rapid genome recoding by iterative recombineering of synthetic DNA

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

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bioRxiv2017

Daisy quorum drives for the genetic restoration of wild populations

An ideal gene drive system to alter wild populations would 1) exclusively affect organisms within the political boundaries of consenting communities, and 2) be capable of restoring any engineered population to its original genetic state. Here we describe daisy quorum drive systems that meet these criteria by combining daisy drive with underdominance. A daisy quorum drive system is predicted to spread through a population until all of its daisy elements have been lost, at which point its fitness becomes frequency-dependent: mostly altered populations become fixed for the desired change, while engineered genes at low frequency are swiftly eliminated by natural selection. The result is an engineered population surrounded by wild-type organisms with limited mixing at the boundary. Releasing large numbers of wild-type organisms or a few bearing a population suppression element can reduce the engineered population below the quorum, triggering elimination of all engineered sequences. In principle, the technology can restore any drive-amenable population carrying engineered genes to wild-type genetics. Daisy quorum systems may enable efficient, community-supported, and genetically reversible ecological engineering.\n\nSummaryLocal communities should be able to control their own environments without forcing those choices on others. Ideally, each community could reversibly alter local wild organisms in ways that cannot spread beyond their own boundaries, and any engineered population could be restored to its original genetic state. We've invented a 'daisy quorum' drive system that appears to meet these criteria.\n\n\"Daisy\" refers to a daisy drive, which typically uses a daisy-chain of linked genes to spread a change through a local population while losing links every generation until it stops spreading. \"Quorum\" reflects the system's ability to \"vote\" on whether a local population should be altered or not: once all daisy elements are lost, it favors replication by the altered version or the original depending on which is more abundant in the local area. Put together, they result in a change that first spreads through a local population, then either becomes locally prevalent is eliminating, inhibiting mixing at the boundary. All organisms in the target population are altered, but changes are unable to spread much beyond that area due to being greatly outnumbered by wild-type organisms and consequently less able to replicate.\n\nWe haven't yet performed any experiments involving daisy quorum systems. Rather, were describing what we intend to do, including the safeguards we will use and our assessment of risks, in the hope that others will evaluate our plans and tell us if there's anything wrong that we missed. We hope that all researchers working on gene drive systems - and other technologies that could impact the shared environment - will similarly pre-register their plans. Sharing plans can reduce needless duplication, accelerate progress, and make the proposed work safer for everyone.

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bioRxiv2017

Intracellular production of hydrogels and synthetic RNA granules by multivalent enhancers

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.

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bioRxiv2017

Bacterial glycoengineering as a biosynthetic route to customized glycomolecules

Bacteria have garnered increased interest in recent years as a platform for the biosynthesis of a variety of glycomolecules such as soluble oligosaccharides, surface-exposed carbohydrates and glycoproteins. The ability to flexibly engineer commonly used laboratory species such as Escherichia coli to efficiently synthesize non-native sugar structures by recombinant expression of enzymes from various carbohydrate biosynthesis pathways has allowed for the facile generation of important products such as conjugate vaccines, glycosylated outer membrane vesicles, and a variety of other research reagents for studying and understanding the role of glycans in living systems. This chapter highlights some of the key discoveries and technologies for equipping bacteria with the requisite biosynthetic machinery to generate such products. As the bacterial glyco-toolbox continues to grow, these technologies are expected to expand the range of glycomolecules produced recombinantly in bacterial systems, thereby opening up this platform to an even larger number of applications.

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bioRxiv2017

High-Throughput Characterization of Protein-Protein Interactions by Reprogramming Yeast Mating

High-throughput methods for screening protein-protein interactions (PPIs) enable the rapid characterization of engineered binding proteins and interaction networks. While existing methods are powerful, none allow quantitative library-on-library characterization of PPIs in a modifiable extracellular environment. Here, we show that sexual agglutination of S. cerevisiae can be reprogrammed to link PPI strength with mating efficiency using yeast synthetic agglutination (YSA). Validation of YSA with 96 previously characterized interactions shows a strong log-linear relationship between mating efficiency and PPI strength for interactions with KDs ranging from 500 pM to 25 M. Using induced chromosomal translocation to pair barcodes representing interacting proteins, thousands of distinct interactions can be screened in a single pot. YSA binding interactions occur in a controllable extracellular environment, and thus studying the effects of environmental factors on PPI networks is possible. YSA enables the high-throughput, quantitative characterization of PPI networks in a fully defined extracellular environment at a library-on-library scale.

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bioRxiv2017

Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and unbiased, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple one-pot protocol.

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bioRxiv2017

Spatial gene drives and pushed genetic waves

Gene drives have the potential to rapidly replace a harmful wild-type allele with a gene drive allele engineered to have desired functionalities. However, an accidental or premature release of a gene drive construct to the natural environment could damage an ecosystem irreversibly. Thus, it is important to understand the spatiotemporal consequences of the super-Mendelian population genetics prior to potential applications. Here, we employ a reaction-diffusion model for sexually reproducing diploid organisms to study how a locally introduced gene drive allele spreads to replace the wild-type allele, even though it posses a selective disadvantage s > 0. Using methods developed by N. Barton and collaborators, we show that socially responsible gene drives require 0.5 < s < 0.697, a rather narrow range. In this \"pushed wave\" regime, the spatial spreading of gene drives will be initiated only when the initial frequency distribution is above a threshold profile called \"critical propagule\", which acts as a safeguard against accidental release. We also study how the spatial spread of the pushed wave can be stopped by making gene drives uniquely vulnerable (\"sensitizing drive\") in a way that is harmless for a wild-type allele. Finally, we show that appropriately sensitized drives in two dimensions can be stopped even by imperfect barriers perforated by a series of gaps.

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bioRxiv2017

The WPRE Improves Genetic Engineering With Site-Specific Nucleases

AbstractInclusion of the woodchuck hepatitis virus post-transcriptional response element (WPRE) in the 3 UTR of mRNA encoding zinc-finger or TALE nucleases results in up to a fifty-fold increase in nuclease expression and a several-fold increase in nuclease-modified chromosomes. Significantly, this increase is additive with the enhancement generated by transient hypothermic shock. The WPRE-mediated improvement is seen across several types of human and mouse primary and transformed cells and is translatable in vivo to the mouse liver.

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