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Results for “Synthetic Biology”

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bioRxiv2018

A Bacterial Expression Vector Archive (BEVA) forflexible modular assembly of Golden Gate-compatible vectors

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. To demonstrate the potential of the system three example vectors were constructed and tested. Golden Gate level 1 vectors; pOGG024, with a broad-host range and high copy number was used for gene expression in laboratory-cultured Rhizobium leguminosarum, and pOGG026, with a broad-host range a lower copy number and excellent stability, even in the absence of antibiotic selection. The application of pOGG026 is demonstrated in environmental samples by bacterial gene expression in nitrogen-fixing nodules on pea plants roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, for which we demonstrate an application by constructing a dual reporter plasmid expressing green and red fluorescent proteins.\n\nIMPORTANCEModular assembly is powerful as it allows easy combining of different components from a library of parts. In designing a modular vector assembly system, the key constituent parts (and modules) are; an origin of plasmid replication, antibiotic resistance marker(s), cloning site(s), together with additional accessory modules as required. In an ideal vector, the size of each module would be minimized, and this we have addressed. We have designed such a vector assembly system by utilizing the Type IIS restriction enzyme Esp3I and have demonstrated its use for Golden Gate cloning in Escherichia coli. An important attribute of this modular vector assembly is that using the principles outlined here, new modules for specific applications, e.g. origin of replication for plasmids in other bacteria, can easily be designed. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach.

microbiology↗

bioRxiv2018

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the shape index, a characteristic feature of each TSS describing the spatial distribution of transcription initiation events, has a surprisingly strong negative correlation with the measured expression levels. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

systems biology↗

bioRxiv2018

Environmental disturbances compromise synthetic symbiosis

By virtue of complex interactions, the behaviour of mutualistic systems is difficult to study and nearly impossible to predict. We have developed a theoretical model of a modifiable experimental yeast system that is amenable to exploring self-organised cooperation while considering the production and use of specific metabolites. Leveraging the simplicity of an artificial yeast system, a simple model of mutualism, we develop and test the assumptions and stability of this theoretical model. We examine how one-off, recurring and permanent changes to an ecological niche affect a cooperative interaction and identify an ecological \"Goldilocks zone\" in which the mutualism can survive. Moreover, we explore how a factor like the cost of mutualism - the cellular burden of cooperating - influences the stability of mutualism and how environmental changes shape this stability. Our results highlight the fragility of mutualisms and suggest the use of synthetic biology to stave off an ecological collapse.

evolutionary biology↗

bioRxiv2018

Catalytic promiscuity potentiated the divergence of new cytochrome P450 enzyme functions in cyanogenic defense metabolism

Cytochrome P450 monooxygenases (P450s) constitute the largest metabolic enzyme family in plants, responsible for synthesizing hundreds of thousands of specialized metabolites with essential roles in chemical defenses against herbivores and pathogens (Banks et al., 2011; Nelson and Werck-Reichhart, 2011; Wurtzel and Kutchan, 2016). Substrate promiscuity has been documented to play a central role in the evolution of plant specialized metabolic enzymes (Weng et al., 2012; Leong and Last, 2017), however most plant P450s are highly substrate-specific (Verpoorte, 2013). Here, we show the rapid inversion of primary and weak secondary (promiscuous) catalytic activities between two distinct yet evolutionarily linked multifunctional P450s, CYP71A12 and CYP71A13, based on intramolecular epistasis of two amino acid residues under positive selection in CYP71A12. Furthermore, we uncover previously undocumented catalytic activity during the inversion as well as naturally occurring amino acid substitution patterns that could have been present in evolutionary intermediates between the two enzymes. Comparative expression profiling and homology modeling reveal that natural selection acted on the promoter of CYP71A13 and the substrate-recognition elements of CYP71A12 to improve the efficiencies of their promiscuous reactions. The rise in catalytic promiscuity potentiated the divergence of new P450 enzyme functions in cyanogenic defense metabolism. Directed evolution of promiscuous reactions is one of the core technologies underpinning the field of synthetic biology. Our results provide a more complete understanding of how natural selection uses promiscuous reactions to generate new enzymes in nature and chemical diversity in pathogen defense, as well as demonstrate a novel strategy for identifying their molecular origins in highly divergent, related enzymes.

evolutionary biology↗

bioRxiv2018

Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

Cell-free gene expression using purified components or cell extracts has become an important platform for synthetic biology that is finding a growing numBer of practical applications. Unfortunately, at cell-relevant reactor volumes, cell-free expression suffers from excessive variability (noise) such that protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers. Consensus opinion holds that variability in expression is due to the stochastic distribution of expression resources (DNA, RNAP, ribosomes, etc.) across the population of reaction chambers. In contrast, here we find that chamber-to-chamber variation in the expression efficiency generates the large variability in protein production. Through analysis and modeling, we show that chambers self-organize into expression centers that control expression efficiency. Chambers that organize into many centers, each having relatively few expression resources, exhibit high expression efficiency. Conversely, chambers that organize into just a few centers where each center has an abundance of resources, exhibit low expression efficiency. A particularly surprising finding is that diluting expression resources reduces the chamber-to-chamber variation in protein production. Chambers with dilute pools of expression resources exhibit higher expression efficiency and lower expression noise than those with more concentrated expression resources. In addition to demonstrating the means to tune expression noise, these results demonstrate that in cell-free systems, self-organization may exert even more influence over expression than the abundance of the molecular components of transcription and translation. These observations in cell-free platform may elucidate how self-organized, membrane-less structures emerge and function in cells.

biophysics↗

bioRxiv2018

DeepCDpred: Inter-residue Distance and Contact Predictionfor Improved Prediction of Protein Structure.

Rapid, accurate prediction of protein structure from amino acid sequence would accelerate fields as diverse as drug discovery, synthetic biology and disease diagnosis. Massively improved prediction of protein structures has been driven by improving the prediction of the amino acid residues that contact in their 3D structure. For an average globular protein, around 92% of all residue pairs are non-contacting, therefore accurate prediction of only a small percentage of inter-amino acid distances could increase the number of constraints to guide structure determination. We have trained deep neural networks to predict inter-residue contacts and distances. Distances are predicted with an accuracy better than most contact prediction techniques. Addition of distance constraints improved de novo structure predictions for test sets of 158 protein structures, as compared to using the best contact prediction methods alone. Importantly, usage of distance predictions allows the selection of better models from the structure pool without a need for an external model assessment tool. The results also indicate how the accuracy of distance prediction methods might be improved further.

bioinformatics↗

bioRxiv2018

A well-controlled BioID design for endogenous bait proteins.

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate overexpression of bait proteins. Control cell lines are typically non-engineered cells or engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also transform BioID, a proximity labelling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over classical ectopic expression of p53-BirA*, and we provide a first well-controlled view on the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC=\"FIGDIR/small/427807v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@404da1org.highwire.dtl.DTLVardef@1a8af36org.highwire.dtl.DTLVardef@18741a6org.highwire.dtl.DTLVardef@e73ab3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation

The CRISPR/Cas9 system has made significant contribution to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems and disease conditions require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current co-transfection based sgRNA co-expression systems remain poorly efficient and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single non-repetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its strong potency, our method will greatly facilitate the understandings in genome structure, function and dynamics, and will contribute to the systemic investigations into complex physiological and pathological conditions.

synthetic biology↗

bioRxiv2017

A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC=\"FIGDIR/small/109306_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@9d07d1org.highwire.dtl.DTLVardef@16c6a41org.highwire.dtl.DTLVardef@b3e6a6org.highwire.dtl.DTLVardef@eba2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2014

An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells

RNA-based regulation, such as RNA interference, and CRISPR/Cas transcription factors (CRISPR-TFs), can enable scalable synthetic gene circuits and the modulation of endogenous networks but have yet to be integrated together. Here, we combined multiple mammalian RNA regulatory strategies, including RNA triple helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing in human cells. We describe three complementary strategies for expressing functional gRNAs from transcripts generated by RNA polymerase II (RNAP II) promoters while allowing the harboring gene to be translated. These architectures enable the multiplexed expression of proteins and multiple gRNAs from a single compact transcript for efficient modulation of synthetic constructs and endogenous human promoters. We used these regulatory tools to implement tunable synthetic gene circuits, including multi-stage transcriptional cascades. Finally, we show that Csy4 can rewire regulatory connections in RNA-dependent gene circuits with multiple outputs and feedback loops to achieve complex functional behaviors. This multiplexable toolkit will be valuable for the construction of scalable gene circuits and the perturbation of natural regulatory networks in human cells for basic biology, therapeutic, and synthetic-biology applications.

Synthetic Biology↗

bioRxiv2014

In vivo generation of DNA sequence diversity for cellular barcoding

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic \"barcode\" that can be recovered by DNA sequencing. We demonstrate the feasibility of this technique in bacterial cells. This method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples.

Synthetic Biology↗

bioRxiv2016

Rapidly evolving homing CRISPR barcodes

We present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cultured cell populations and show that they can record lineage history and and that their RNA can be assayed as single molecules in situ. This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

Synthetic Biology↗

bioRxiv2014

State-dependent information processing in gene regulatory networks

Cellular information processing is generally attributed to the complex networks of genes and proteins that regulate cell behavior. It is still unclear, however, what are the main features of those networks that allow a cell to encode and interpret its ever changing environment. Here we address this question by studying the computational capabilities of the transcriptional regulatory networks of five evolutionary distant organisms. We identify in all cases a cyclic recurrent structure, formed by a small core of genes, that is essential for dynamical encoding and information integration. The recent history of the cell is encoded by the transient dynamics of this recurrent reservoir of nodes, while the rest of the network forms a readout layer devoted to decode and interpret the high-dimensional dynamical state of the recurrent core. This separation of roles allows for the integration of temporal information, while facilitating the learning of new environmental conditions and preventing catastrophic interference between those new inputs and the previously stored information. This resembles the reservoir-computing paradigm recently proposed in computational neuroscience and machine learning. Our results reveal that gene regulatory networks act as echo-state networks that perform optimally in standard memory-demanding tasks, and confirms that most of their memory resides in the recurrent reservoir. We also show that the readout layer can learn to decode the information stored in the reservoir via standard evolutionary strategies. Our work thus suggests that recurrent dynamics is a key element for the processing of complex time-dependent information by cells.\n\nSummaryCells must monitor the dynamics of their environment continuously, in order to adapt to present conditions and anticipate future changes. But anticipation requires processing temporal information, which in turn requires memory. Here we propose that cells can perform such dynamical information processing via the reservoir computing paradigm. According to this concept, a structure with recurrent (cyclic) paths, known as the reservoir, stores in its dynamics a record of the cells recent history. A much simpler feedforward structure then reads and decodes that information. We show that the transcriptional gene regulatory networks of five evolutionary distant organisms are organized in this manner, allowing them to store complex time-dependent signals entering the cell in a biologically realistic manner.

Synthetic Biology↗

bioRxiv2018

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

synthetic biology↗

bioRxiv2018

Reproducing bench-scale cell growth and productivity

Reproducing, exchanging, comparing, and building on each others work is foundational to technology advances.1 Advancing biotechnology calls for reliable reuse of engineered strains.2 Reliable reuse of engineered strains requires reproducible growth and productivity. To demonstrate reproducibility for biotechnology, we identified the experimental factors that have the greatest effect on the growth and productivity of our engineered strains.3-6 We present a draft of a Minimum Information Standard for Engineered Organism Experiments (MIEO) based on this method. We evaluated the effect of 22 factors on Escherichia coli (E. coli) engineered to produce the small molecule lycopene, and 18 factors on E. coli engineered to produce red fluorescent protein (RFP). Container geometry and shaking had the greatest effect on product titer and yield. We reproduced our results under two different conditions of reproducibility:7 conditions of use (different fractional factorial experiments), and time (48 biological replicates performed on 12 different days over four months).

synthetic biology↗

bioRxiv2018

Ultrasensitive molecular controllers for quasi-integral feedback

Feedback control has enabled the success of automated technologies by mitigating the effects of variability, unknown disturbances, and noise. Similarly, feedback loops in biology reduce the impact of noise and help shape kinetic responses, but it is still unclear how to rationally design molecular controllers that approach the performance of controllers in traditional engineering applications, in particular the performance of integral controllers. Here, we describe a strategy to build molecular quasi-integral controllers by following two design principles: (1) a highly ultrasensitive response, which guarantees a small steady-state error, and (2) a tunable ultrasensitivity threshold, which determines the system equilibrium point (reference). We describe a molecular reaction network, which we name Brink motif, that satisfies these requirements by combining sequestration and an activation/deactivation cycle. We show that if ultrasensitivity conditions are satisfied, this motif operates as a quasi-integral controller and promotes homeostatic behavior of the closed-loop system (robust tracking of the input reference while rejecting disturbances). We propose potential biological implementations of Brink controllers and we illustrate different example applications with computational models.

synthetic biology↗

bioRxiv2017

Gene synthesis allows biologists to source genes from farther away in the tree of life

Gene synthesis allows biologists and bioengineers to create novel genetic sequences and codon-optimize transgenes for heterologous expression. Because codon choice is key to gene expression and therefore cellular outcomes, it has been argued that gene synthesis will allow researchers to source genes from organisms that would otherwise have been incompatible, opening up more-distant parts of the tree of life as sources for transgenes. We test if this hypothesis is true for academic biological research using a 10-year data set from Addgene, the non-profit plasmid repository. We observe ~19,000 unique genes deposited to Addgene and classify them by whether they are natural or synthetic using a nucleotide-only technique that we develop here. We find that synthetic genes are an increasing share of Addgene deposits. Most importantly, we find direct evidence that researchers are using gene synthesis to source genes from more genetically-distant organisms, particularly for genes that are longer and thus might otherwise be particularly challenging to express. Thus, we provide the first empirical evidence that gene synthesis is leading biologists and bioengineers to sample more broadly across the rich genetic diversity of life, increasingly making that functionality available for industrial or biomedical advances.

synthetic biology↗

bioRxiv2017

Redesigning chromosomes to optimize conformation capture (Hi-C) assays

In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous, large identical regions remain invisible to the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-3C design, we enhanced the signal to noise ratio and improved the visibility of the entire region. We also improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

synthetic biology↗