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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 65,085 bioRxiv papers from 288,430 authors.

Most downloaded bioRxiv papers, all time

in category synthetic biology

607 results found. For more information, click each entry to expand.

581: TFound: A Software to Map Cis-Regulatory Elements in Yeast
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Posted to bioRxiv 23 Nov 2018

TFound: A Software to Map Cis-Regulatory Elements in Yeast
164 downloads synthetic biology

Adriano Gomes-Silva, Rafael Silva-Rocha

Transcription factors (TFs) are responsible for regulating the rate of transcription of genes in all organisms. These factors can be represented computationally by position weight matrices (PWM). TFound was developed to allow the detailed visualization of predicted binding sites of transcription factors in multiple sequences based on the PWMs, by using the graphic user interface (GUI). The tool was loaded with the genome of Saccharomyces cerevisiae and PWMs from the YeTFaSCo database (http://yetfasco.ccbr.utoronto.ca/), also allowing the insertion of new sequences and PWMs. Thus, the user is allowed to load custom PWMs and genomes to perform easily mining and visualization of binding site motifs of cis-regulatory elements of interest, permitting an efficient way to inspect DNA assembly projects for complex synthetic circuits. This work describes the functionality of the current version of the tool, which is coded in Python and is freely available at the repository https://github.com/adri4nogomes/TFound.

582: Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro
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Posted to bioRxiv 15 May 2018

Expression of novel fusion antiviral proteins Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro
162 downloads synthetic biology

Yasser S. Hassan, Sherry Ogg, Hui Ge

Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-PAPS1 was produced in E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with 50% protein synthesis inhibitory concentration (IC50) of 0.06nM (3.63ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with an IC50 of 0.03nM (1.82ng/ml) and a therapeutic index of >21818. The fusion protein was further optimized using in silico tools, produced in E. coli in vivo expression system, purified by three-step process from soluble lysate and protein synthesis inhibition activity assayed. Results showed that the optimized protein (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function activity on protein synthesis inhibition with an IC50 of 0.03nM (1.82ng/ml). Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant antiviral activity against HBV in vitro with a high therapeutic index and, thus, meriting further development as potential antiviral agents against chronic HBV infections.

583: Securing the exchange of synthetic genetic constructs using digital signatures
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Posted to bioRxiv 26 Sep 2019

Securing the exchange of synthetic genetic constructs using digital signatures
162 downloads synthetic biology

Jenna E Gallegos, Diptendu M Kar, Indrakshi Ray, Indrajit Ray, Jean Peccoud

Synthetic biologists use a growing number of software tools to generate DNA sequences encoding complex functions. In this context, some synthetic biologists have inserted watermarks in synthetic DNA to assert claims of authorships. DNA watermarking demonstrates the need to assert the rights and responsibilities associated with authorships of synthetic sequences. However, watermarks lack the properties necessary to secure the exchange of synthetic genetic material. In this manuscript, we describe how data encryption and digital signature algorithms can be used to ensure the integrity and authenticity of synthetic genetic constructs. We demonstrate that for 91/96 isolates, we can predictably extract information about the author, the identity, and the integrity of plasmid sequences from sequencing data alone without a reference, all without compromising the function of the plasmids. We discuss how this technology can be improved, applied, and expanded to support trustworthy transactions in the bioeconomy supply chain.

584: Engineering a functional NifDK polyprotein resistant to mitochondrial degradation
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Posted to bioRxiv 03 Sep 2019

Engineering a functional NifDK polyprotein resistant to mitochondrial degradation
162 downloads synthetic biology

Robert S Allen, Christina M Gregg, Shoko Okada, Amratha Menon, Dawar Hussain, Vanessa Gillepsie, Ema Johnston, Andrew C Warden, Matthew Taylor, Michelle Colgrave, Keren Byrne, Craig Wood

To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

585: Potentiating antibiotic treatment using fitness-neutral gene expression perturbations
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Posted to bioRxiv 15 Aug 2019

Potentiating antibiotic treatment using fitness-neutral gene expression perturbations
159 downloads synthetic biology

Peter B Otoupal, Keesha E Erickson, Kristen A Eller, Jocelyn K Campos, Thomas R Aunins, Anushree Chatterjee

The rapid proliferation of multidrug-resistant (MDR) bacteria poses a critical threat to human health, for which new antimicrobial strategies are desperately needed. Here we outline a strategy for combating bacterial infections by administering fitness neutral gene expression perturbations as co-therapies to potentiate antibiotic lethality. We systematically explored the fitness of 270 gene knockout-drug combinations in Escherichia coli, identifying 114 synergistic interactions. Genes revealed in this screen were subsequently perturbed at the transcriptome level via multiplexed CRISPR-dCas9 interference to induce antibiotic synergy. These perturbations successfully sensitized E. coli to antibiotic treatment without imposing a separate fitness cost. We next administered these fitness neutral gene perturbations as co-therapies to potentiate antibiotic killing of Salmonella enterica in intracellular infections of HeLa epithelial cells, demonstrating therapeutic applicability. Finally, we utilized these results to design peptide nucleic acid (PNA) co-therapies for targeted gene expression reduction in four MDR, clinically isolated bacteria. Two isolates of Klebsiella pneumoniae and E. coli were each exposed to PNAs targeting homologs of the genes csgD, fnr, recA and acrA in the presence of sub-minimal inhibitory concentrations of trimethoprim. We successfully increased each strains susceptibility to trimethoprim treatment and identified eight cases in which re-sensitization occurred without a direct fitness impact of the PNA on MDR strains. Our results highlight a promising approach for combating MDR bacteria which could extend the utility of our current antibiotic arsenal.

586: Maximum Caliber can build and infer models of oscillation in a three-gene feedback network
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Posted to bioRxiv 16 Oct 2018

Maximum Caliber can build and infer models of oscillation in a three-gene feedback network
159 downloads synthetic biology

Taylor Firman, Anar Amgalan, Kingshuk Ghosh

Single-cell protein expression time trajectories provide rich temporal data quantifying cellular variability and its role in dictating fitness. However, theoretical models to analyze and fully extract information from these measurements remain limited for three reasons: i) gene expression profiles are noisy, rendering models of averages inapplicable, ii) experiments typically measure only a few protein species while leaving other molecular actors -- necessary to build traditional bottom-up models -- unnoticed, and iii) measured data is in fluorescence, not particle number. We have recently addressed these challenges in an alternate top-down approach using the principle of Maximum Caliber (MaxCal) to model genetic switches with one and two protein species. In the present work we address scalability and broader applicability of MaxCal by extending to a three-gene (A, B, C) feedback network that exhibits oscillation, commonly known as the repressilator. We test MaxCal's inferential power by using synthetic data of noisy protein number time traces -- serving as a proxy for experimental data -- generated from a known underlying model. We notice that the minimal MaxCal model -- accounting for production, degradation, and only one type of symmetric coupling between all three species -- reasonably infers several underlying features of the circuit such as the effective production rate, degradation rate, frequency of oscillation, and protein number distribution. Next, we build models of higher complexity including different levels of coupling between A, B, and C and rigorously assess their relative performance. While the minimal model (with four parameters) performs remarkably well, we note that the most complex model (with six parameters) allowing all possible forms of crosstalk between A, B, and C slightly improves prediction of rates, but avoids ad-hoc assumption of all the other models. It is also the model of choice based on Bayesian Information Criteria. We further analyzed time trajectories in arbitrary fluorescence (using synthetic trajectories) to mimic realistic data. We conclude that even with a three-protein system including both fluorescence noise and intrinsic gene expression fluctuations, MaxCal can faithfully infer underlying details of the network, opening up future directions to model other network motifs with many species.

587: Investigating the long-term stability of protein immunogen(s) for whole recombinant yeast-based vaccines
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Posted to bioRxiv 08 Jun 2018

Investigating the long-term stability of protein immunogen(s) for whole recombinant yeast-based vaccines
158 downloads synthetic biology

Ravinder Kumar

Even today vaccine(s) remains a mainstay in combating infectious diseases. Many yeast-based vaccines are currently in different phases of clinical trials. Despite the encouraging results of whole recombinant yeast (WRY) and yeast display (YD), the systematic study assessing the long-term stability of protein antigen(s) in yeast cells is still missing. Therefore, in the present study, I investigate the stability of heterologous protein antigen in the cellular environment of S. cerevisiae through E. coli surface protein (major curlin or CsgA). Present biochemical data showed that the stationary phase yeast cells were able to keep the antigen stable for almost one year when stored at 2-8°C and 23-25°C. Further, iTRAQ based quantitative proteomics of yeast whole cell lysate showed that the level of heterologous fusion protein was low in cells stored at 23-25°C compared to those at 2-8°C. In the end, I also proposed a workable strategy to test integrity or completeness of heterologous protein in the yeast cell. I believe that the observations made in the present study will be really encouraging for those interested in the development of a whole recombinant yeast-based vaccine(s).

588: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
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Posted to bioRxiv 01 Sep 2019

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
156 downloads synthetic biology

Gabriel A Suarez, Kyle R Dugan, Brian A Renda, Sean P Leonard, Lakshmi S Gangavarapu, Jeffrey E. Barrick

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

589: Mathematical modelling of serine integrase - mediated gene assembly
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Posted to bioRxiv 02 Oct 2018

Mathematical modelling of serine integrase - mediated gene assembly
156 downloads synthetic biology

Alexandra Pokhilko, Steven Kane, W. Marshall Stark, Sean D. Colloms

Site-specific recombination promoted by serine integrases can be used for ordered assembly of DNA fragments into larger arrays. When a plasmid vector is included in the assembly, the circular product DNA molecules can transform E. coli cells. A convenient one-pot method using a single integrase involves recombination between pairs of matched orthogonal attachment sites, allowing assembly of up to six DNA fragments. However, the efficiency of assembly decreases as the number of fragments increases, due to accumulation of incorrect products in which recombination has occurred between mismatched sites. Here we use mathematical modelling to analyse published experimental data for the assembly reactions and suggest potential ways to improve assembly efficiency. We assume that unproductive synaptic complexes between pairs of mismatched sites become predominant as the number and diversity of sites increase. Our modelling predicts that the proportion of correct products can be improved by raising fragment DNA concentrations and lowering plasmid vector concentration. The assembly kinetics is affected by the inactivation of integrase in vitro. The model also predicts that the precision might be improved by redesigning the location of attachment sites on fragments to reduce the formation of the wrong circular products. Our preliminary experimental explorations of assembly with fC31 integrase confirmed that assembly efficiency might be improved. However, optimization of efficiency would require more experimental work on the mechanisms of wrong product formation. The use of a more efficient integrase (such as Bxb1) might be a more promising approach to assembly optimization. The model might be easily extended for different integrases or/and different assembly strategies, such as those using multiple integrases or multiple substrate structures.

590: A comprehensive conceptual and computational dynamics framework for Autonomous Regeneration Systems
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Posted to bioRxiv 28 Oct 2019

A comprehensive conceptual and computational dynamics framework for Autonomous Regeneration Systems
155 downloads synthetic biology

Tran Nguyen Minh-Thai, Sandhya Samarasinghe, Michael Levin

This paper presents a new conceptual and computational dynamics framework for damage detection and regeneration in multicellular structures similar to living animals. The model uniquely achieves complete and accurate regeneration from any damage anywhere in the system. We demonstrated the efficacy of the proposed framework on an artificial organism consisting of three tissue structures corresponding to the head, body and tail of a worm. Each structure consists of a stem cell surrounded by a tissue of differentiated cells. We represent a tissue as an Auto-Associative Neural Network (AANN) with local interactions and stem cells as a self-repair network with long-range interactions. We also propose another new concept, Information Field which is a mathematical abstraction over traditional components of tissues, to keep minimum pattern information of the tissue structures to be accessed by stem cells in extreme cases of damage. Through entropy, a measure of communication between a stem cell and differentiated cells, stem cells monitor the tissue pattern integrity, violation of which triggers damage detection and tissue repair. Stem cell network monitors its state and invokes stem cell repair in the case of stem cell damage. The model accomplishes regeneration at two levels: In the first level, damaged tissues with intact stem cells regenerate themselves. Here, stem cell identifies entropy change and finds the damage and regenerates the tissue in collaboration with the AANN. In the second level, involving missing whole tissues and stem cells, the remaining stem cell(s) access the information field to restore the stem cell network and regenerate missing tissues. In the case of partial tissue damage with missing stem cells, the two levels collaborate to accurately restore the stem cell network and tissues. This comprehensive hypothetical framework offers a new way to conceptualise regeneration for better understanding the regeneration processes in living systems. It could also be useful in biology for regenerative medicine and in engineering for building self-repairing biobots.

591: Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum
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Posted to bioRxiv 04 Sep 2019

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum
150 downloads synthetic biology

Heongmin Seo, Jong-Won Lee, Sergio Garcia, Cong T. Trinh

Background: Esters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows thermophilic production with advantageous downstream product separation. However, due to the limited thermal stability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for thermophilic ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum. Results: In this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperature. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated the engineered C. thermocellum could produce isobutyl acetate directly from cellulose. Conclusions: This study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

592: Harnessing natural modularity of cellular metabolism to design a modular chassis cell for a diverse class of products by using goal attainment optimization
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Posted to bioRxiv 28 Aug 2019

Harnessing natural modularity of cellular metabolism to design a modular chassis cell for a diverse class of products by using goal attainment optimization
150 downloads synthetic biology

Garcia Sergio, Cong T. Trinh

Living cells optimize their fitness against constantly changing environments to survive. Goal attainment optimization is a mathematical framework to describe the simultaneous optimization of multiple conflicting objectives that must all reach a performance above a threshold or goal. In this study, we applied goal attainment optimization to harness natural modularity of cellular metabolism to design a modular chassis cell for optimal production of a diverse class of products, where each goal corresponds to the minimum biosynthesis requirements (e.g., yields and rates) of a target product. This modular cell design approach enables rapid generation of optimal production strains that can be assembled from a modular cell and various exchangeable production modules and hence accelerates the prohibitively slow and costly strain design process. We formulated the modular cell design problem as a blended or goal attainment mixed integer linear program, using mass-balance metabolic models as biological constraints. By applying the modular cell design framework for a genome-scale metabolic model of Escherichia coli, we demonstrated that a library of biochemically diverse products could be effectively synthesized at high yields and rates from a modular (chassis) cell with only a few genetic manipulations. Flux analysis revealed this broad modularity phenotype is supported by the natural modularity and flexible flux capacity of core metabolic pathways. Overall, we envision the developed modular cell design framework provides a powerful tool for synthetic biology and metabolic engineering applications such as industrial biocatalysis to effectively produce fuels, chemicals, and therapeutics from renewable and sustainable feedstocks, bioremediation, and biosensing.

593: Incorporating Randomness into DNA Steganography to Realize Secondary Secret key, Self-destruction, and Quantum Key Distribution-like Function
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Posted to bioRxiv 05 Aug 2019

Incorporating Randomness into DNA Steganography to Realize Secondary Secret key, Self-destruction, and Quantum Key Distribution-like Function
136 downloads synthetic biology

Meiying Cui, Yixin Zhang

DNA has become a promising candidate as a future data storage medium, which makes DNA steganography indispensable in DNA data security. While PCR primers are conventional secret keys in DNA steganography, the information can be read once the primers are intercepted. New steganography approach is needed to make the DNA-encoded information safer, if not unhackable. Herein, by mixing information-carrying DNA with partially degenerated DNA library containing single or multiple restriction sites, we build an additional protective layer, which can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA-encrypted information, heating will cause reshuffling and generate endonuclease-resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, the DNA steganography possesses both quantum key distribution (QKD)-like function for detecting PCR by an interceptor and self-destructive property. With a DNA-ink incorporating the steganography, the authenticity of writing can be confirmed only by an authorized person with the knowledge of all embedded keys.

594: Chemical Characterization of Interacting Genes in Few Subnetworks of Alzheimer's Disease
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Posted to bioRxiv 11 Jul 2018

Chemical Characterization of Interacting Genes in Few Subnetworks of Alzheimer's Disease
133 downloads synthetic biology

Antara Sengupta, Hazel Nicolette Manners, Pabitra Pal Choudhury, Swarup Roy

A number of genes have been identified as a key player in Alzheimers disease (AD). Topological analysis of co-expression network reveals that key genes are mostly central or hub genes. The association between a hub gene and its neighbour genes can be derived easily using relative abundance of their expression levels. However, it is still unexplored fact that whether any hub and its neighbour genes within a subnetwork exhibits any kind of proximity with respect to their chemical properties of the DNA sequences or not, that code for a sequence of amino acids. In this work, we try to make a quantitative investigation of the underlying biological facts in DNA sequential and primary protein level in mathematical paradigm. It may gives a holistic view of the interrelationships existing between hub genes and neighbour genes in few selective AD subnetworks. We define a mapping model from physicochemical properties of DNA sequence to chemical characterization of amino acid sequences. We use distribution of chemical groups present in a sequence after decoding into corresponding amino acids to investigate the fact that whether any hub genes are associated closely with its neighbour genes chemically in the subnetworks. Interestingly, our preliminary results confirm the fact the dependent genes that are coexpressed with its hub gene are also having proximity with respect to their amino acid chemical group distributions.

595: Biosynthesis of alkylcitric acids in Aspergillus niger involves both co-localized and unlinked genes
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Posted to bioRxiv 25 Jul 2019

Biosynthesis of alkylcitric acids in Aspergillus niger involves both co-localized and unlinked genes
130 downloads synthetic biology

Sylvester Palys, Thi Thanh My Pham, Adrian Tsang

Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl tail and a citrate-derived head. We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from a litre of culture filtrate 8.5 grams of extract containing seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised ~ 95% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed an in-cluster cis-aconitate decarboxylase gene (cadA) in A. oryzae and A. flavus, which in A. niger is located on a different chromosome. Overexpression of the A. niger cadA and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

596: Experimental Evidence for Constraints in Amplitude-Timescale Co-variation of a Biomolecular Pulse Generating Circuit Design
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Posted to bioRxiv 03 Sep 2019

Experimental Evidence for Constraints in Amplitude-Timescale Co-variation of a Biomolecular Pulse Generating Circuit Design
130 downloads synthetic biology

Abhilash Patel, Shaunak Sen

Understanding constraints on the functional properties of biomolecular circuit dynamics, such as the variation of amplitude and timescale of pulse, is an important part of biomolecular circuit design. While the amplitude-timescale co-variations of the pulse in an incoherent feedforward loop have been investigated computationally using mathematical models, experimental support for such constraints is relatively unclear. Here, we address this using experimental measurements of an existing pulse generating incoherent feedforward loop circuit realization in the context of a standard mathematical model. We characterize the trends of co-variation in the pulse amplitude and rise time computationally by randomly exploring the parameter space. We experimentally measured the co-variation by varying inducers and found that larger amplitude pulses have slower rise time. We discuss the gap between the experimental measurements and predictions of the standard model, highlighting model additions and other biological factors that might bridge the gap.

597: Assessment of Robustness to Temperature in a Negative Feedback Loop and a Feedforward Loop
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Posted to bioRxiv 18 Sep 2019

Assessment of Robustness to Temperature in a Negative Feedback Loop and a Feedforward Loop
126 downloads synthetic biology

Abhilash Patel, Richard M. Murray, Shaunak Sen

Robustness to temperature variation is an important specification for biomolecular circuit design. While cancellation of parametric temperature dependences has been shown to improve temperature robustness of period in a synthetic oscillator design, the performance of other biomolecular circuit designs in different temperature conditions is relatively unclear. Using a combination of experimental measurements and mathematical models, we assess the temperature robustness of two biomolecular circuit motifs \---| a negative feedback loop and a feedforward loop. We find that the measured responses in both circuits can change with temperature, both in the amplitude and in the transient response. We find that, in addition to the cancellation of parametric temperature dependencies, certain parameter regimes can also facilitate temperature robustness for the negative feedback loop, although at a performance cost. We discuss these parameter regimes of operation in the context of the measured data for the negative feedback loop. These results should help develop a framework for assessing and designing temperature robustness in biomolecular circuits.

598: Enzyme Catalytic Activity Emulated Within DNA-based Nanodevice
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Posted to bioRxiv 16 Oct 2019

Enzyme Catalytic Activity Emulated Within DNA-based Nanodevice
125 downloads synthetic biology

Tadija Kekic, Yasaman Ahmadi, Ivan Barisic

Artificial enzymes hold great potential in the field of biotechnology. We present an approach towards the bottom-up development of an artificial enzyme using the DNA origami technology. A set of peptide-oligonucleotide conjugates, designed to recreate the structure of an active site of a native protein, was placed in the central area of a large, self-folding DNA origami shell structure. The peptide-oligonucleotide conjugates were placed in a predefined positions by the integration on the angle-adjustable, linear constructs that protruded from the nanostructure. We demonstrate a workflow to obtain the essential elements of the protein's active site; to design and assemble the nanodevice; to measure, quantify and inactivate the catalytic activity; and to reuse our structures in multiple experiments. By use of the high-resolution spectroscopy, we demonstrated a significant increase in the product accumulation that originates from the correctly assembled emulated active sites.

599: Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.
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Posted to bioRxiv 28 Oct 2019

Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.
122 downloads synthetic biology

Romel Menacho-Melgar, Zhixia Ye, Eirik A Moreb, Tian Yang, John P Efromson, John S Decker, Michael D Lynch

We report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of 10 grams dry cell weight (gCDW) per liter and protein titers up to 2.7+/-0.2 g/L (270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach ~ 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

600: Effect of diets containing whole or ground corn with and without soybean hulls on feeding calves during the suckling phase
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Posted to bioRxiv 03 Dec 2018

Effect of diets containing whole or ground corn with and without soybean hulls on feeding calves during the suckling phase
113 downloads synthetic biology

Aline Evangelista Machado Santana, José Neuman Miranda Neiva, Vera Lúcia de Araújo Bozorg, João Restle, Ubirajara Bilego

This work evaluated the use of soybean hulls and whole or ground corn in the diets of suckling calves. Diets containing two levels of soybean hull inclusion (0 and 400.1 g/kg) and corn in different physical forms (whole or ground) were evaluated in the diets of newborn crossbred dairy calves that were housed and received experimental diets plus four liters of milk per day over 56 days. Weekly samples of food, diets and leftovers were taken to determine dry matter and nutrient intake. To evaluate the apparent digestibility, samples were taken from the diets, leftovers and feces for three consecutive days using titanium dioxide as an indicator. Blood samples were also collected to evaluate the blood indicators. Including soybean hulls in the diet increased the consumption of neutral detergent fiber but reduced the consumption of non-fibrous carbohydrates, which was also reduced by using whole corn in the diet. Total digestible nutrient consumption did not vary, although its value was reduced by using whole corn and including soybean hulls. The apparent digestibilities of the dry matter and crude protein were similar, resulting in similar performances between the animals, regardless of the factors analyzed. Using soybean hulls or whole corn did not affect blood indicators or feeding costs. Soybean hull or whole corn usage did not affect the performance of crossbred dairy calves during rearing.

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