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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 64,934 bioRxiv papers from 287,775 authors.

Most downloaded bioRxiv papers, all time

in category synthetic biology

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

21: Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery
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Posted to bioRxiv 15 Apr 2018

Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery
3,025 downloads synthetic biology

Suhani Vora, Jenny Cheng, Ru Xiao, Nathan J. VanDusen, Luis Quintino, William T. Pu, Luk H. Vandenberghe, Alejandro Chavez, George Church

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

22: A homing CRISPR mouse resource for barcoding and lineage tracing
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Posted to bioRxiv 12 Mar 2018

A homing CRISPR mouse resource for barcoding and lineage tracing
2,994 downloads synthetic biology

Reza Kalhor, Kian Kalhor, Kathleen Leeper, Amanda Graveline, Prashant Mali, George M Church

Cellular barcoding using nuclease-induced genetic mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We have previously introduced the homing CRISPR system as a promising methodology for generating such barcodes with scalable diversity and without crosstalk. Here, we present a mouse line (MARC1) with multiple genomically-integrated and heritable homing guide RNAs (hgRNAs). We determine the genomic locations of these hgRNAs, their activity profiles during gestation, and the diversity of their mutants. We apply the line for unique barcoding of mouse embryos and differential barcoding of embryonic tissues. We conclude that this mouse line can address the unique challenges associated with in vivo barcoding in mammalian model organisms and is thus an enabling platform for recording and lineage tracing applications in a mammalian model system.

23: An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells
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Posted to bioRxiv 23 Apr 2014

An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells
2,656 downloads synthetic biology

Lior Nissim, Samuel D Perli, Alexandra Fridkin, Pablo Perez-Pinera, Timothy K Lu

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.

24: Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates
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Posted to bioRxiv 01 Mar 2016

Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 NOR gates
2,651 downloads synthetic biology

Miles W Gander, Justin D Vrana, William E. Voje, James M Carothers, Eric Kalvins

Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used genetic components leak transcriptionally, do not allow arbitrary interconnections, or do not have digital responses. Here, we designed a new dCas9-Mxi1 based NOR gate architecture in S. cerevisiae that allows arbitrary connectivity and large genetic circuits. Because we used the strong chromatin remodeler Mxi1, our system showed very little leak and exhibits a highly digital response. In particular, we built a combinatorial library of NOR gates that each directly convert guide RNA (gRNA) input signals into gRNA output signals, enabling NOR gates to be “wired” together. We constructed and characterized logic circuits with up to seven independent gRNAs, including repression cascades with up to seven layers. Modeling predicted that the NOR gates have Hill Coefficients of approximately 1.71±0.09, explaining the minimal signal degradation we observed in these deeply layered circuits. Our approach enables the construction of the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, decision making systems in living cells.

25: Development of prokaryotic cell-free systems for synthetic biology
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Posted to bioRxiv 15 Apr 2016

Development of prokaryotic cell-free systems for synthetic biology
2,619 downloads synthetic biology

Abel C Chiao, Richard M. Murray, Zachary Z Sun

Prokaryotic cell-free systems are currently heavily used for the production of protein that can be otherwise challenging to produce in cells. However, historically cell-free systems were used to explore natural phenomena before the advent of genetic modification and transformation technology. Recently, synthetic biology has seen a resurgence of this historical use of cell-free systems as a prototyping tool of synthetic and natural genetic circuits. For these cell-free systems to be effective prototyping tools, an understanding of cell-free system mechanics must be established that is not purely protein-expression driven. Here we discuss the development of E. coli-based cell-free systems, with an emphasis on documenting published extract and energy preparation methods into a uniform format. We also discuss additional considerations when applying cell-free systems to synthetic biology.

26: Design and analysis of a Proportional-Integral-Derivative controller with biological molecules
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Posted to bioRxiv 18 Apr 2018

Design and analysis of a Proportional-Integral-Derivative controller with biological molecules
2,523 downloads synthetic biology

Michael Chevalier, Mariana Gomez-Schiavon, Andrew Ng, Hana El-Samad

The ability of cells to regulate their function through feedback control is a fundamental underpinning of life. The capability to engineer de novo feedback control with biological molecules is ushering in an era of robust functionality for many applications in biotechnology and medicine. To fulfill their potential, feedback control strategies implemented with biological molecules need to be generalizable, modular and operationally predictable. Proportional-Integral-Derivative (PID) control fulfills this role for technological systems and is a commonly used strategy in engineering. Integral feedback control allows a system to return to an invariant steady-state value after step disturbances, hence enabling its robust operation. Proportional and derivative feedback control used with integral control help sculpt the dynamics of the return to steady-state following perturbation. Recently, a biomolecular implementation of integral control was proposed based on an antithetic motif in which two molecules interact stoichiometrically to annihilate each other's function. In this work, we report how proportional and derivative implementations can be layered on top of this integral architecture to achieve a biochemical PID control design. We illustrate through computational and analytical treatments that the addition of proportional and derivative control improves performance, and discuss practical biomolecular implementations of these control strategies.

27: Evolutionary dynamics of CRISPR gene drives
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Posted to bioRxiv 07 Jun 2016

Evolutionary dynamics of CRISPR gene drives
2,502 downloads synthetic biology

Charleston Noble, Jason Olejarz, Kevin M Esvelt, George M Church, Martin A Nowak

The alteration of wild populations has been discussed as a solution to a number of humanity's most pressing ecological and public health concerns. Enabled by the recent revolution in genome editing, CRISPR gene drives, selfish genetic elements which can spread through populations even if they confer no advantage to their host organism, are rapidly emerging as the most promising approach. But before real-world applications are considered, it is imperative to develop a clear understanding of the outcomes of drive release in nature. Toward this aim, we mathematically study the evolutionary dynamics of CRISPR gene drives. We demonstrate that the emergence of drive-resistant alleles presents a major challenge to previously reported constructs, and we show that an alternative design which selects against resistant alleles greatly improves evolutionary stability. We discuss all results in the context of CRISPR technology and provide insights which inform the engineering of practical gene drive systems.

28: Deep learning enables therapeutic antibody optimization in mammalian cells by deciphering high-dimensional protein sequence space
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Posted to bioRxiv 24 Apr 2019

Deep learning enables therapeutic antibody optimization in mammalian cells by deciphering high-dimensional protein sequence space
2,491 downloads synthetic biology

Derek M Mason, Simon Friedensohn, Cédric R Weber, Christian Jordi, Bastian Wagner, Simon Meng, Pablo Gainza, Bruno E Correia, Sai T Reddy

Therapeutic antibody optimization is time and resource intensive, largely because it requires low-throughput screening (10^3 variants) of full-length IgG in mammalian cells, typically resulting in only a few optimized leads. Here, we use deep learning to interrogate and predict antigen-specificity from a massively diverse sequence space to identify globally optimized antibody variants. Using a mammalian display platform and the therapeutic antibody trastuzumab, rationally designed site-directed mutagenesis libraries are introduced by CRISPR/Cas9-mediated homology-directed repair (HDR). Screening and deep sequencing of relatively small libraries (10^4) produced high quality data capable of training deep neural networks that accurately predict antigen-binding based on antibody sequence. Deep learning is then used to predict millions of antigen binders from an in silico library of ~10^8 variants, where experimental testing of 30 randomly selected variants showed all 30 retained antigen specificity. The full set of in silico predicted binders is then subjected to multiple developability filters, resulting in thousands of highly-optimized lead candidates. With its scalability and capacity to interrogate high-dimensional protein sequence space, deep learning offers great potential for antibody engineering and optimization.

29: Precise Cas9 targeting enables genomic mutation prevention
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Posted to bioRxiv 14 Jun 2016

Precise Cas9 targeting enables genomic mutation prevention
2,444 downloads synthetic biology

Alejandro Chavez, Benjamin W Pruitt, Marcelle Tuttle, Rebecca S Shapiro, Ryan J Cecchi, Jordan Winston, Brian M Turczyk, Michael Tung, James J Collins, George M Church

Here we present a generalized method of guide RNA tuning that enables Cas9 to discriminate between two target sites that differ by a single nucleotide polymorphism. We employ our methodology to generate a novel in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

30: Multiplexed CRISPR-Cas9 based genome editing of Rhodosporidium toruloides
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Posted to bioRxiv 10 Feb 2019

Multiplexed CRISPR-Cas9 based genome editing of Rhodosporidium toruloides
2,402 downloads synthetic biology

Peter B Otoupal, Masakazu Ito, Adam P Arkin, Jon K Magnuson, John M Gladden, Jeffrey Skerker

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9 based gene disruption in R. toruloides. Stably integrating a Cas9 expression cassette into the genome brought about successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high throughput genome engineering in this industrially relevant organism.

31: Assembly of Radically Recoded E. coli Genome Segments
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Posted to bioRxiv 19 Aug 2016

Assembly of Radically Recoded E. coli Genome Segments
2,353 downloads synthetic biology

Julie E. Norville, Cameron L. Gardner, Eduardo Aponte, Conor K. Camplisson, Alexandra Gonzales, David K. Barclay, Katerina A. Turner, Victoria Longe, Maria Mincheva, Jun Teramoto, Kento Tominaga, Ryota Sugimoto, James E. DiCarlo, Marc Guell, Eriona Hysolli, John Aach, Christopher J. Gregg, Barry L. Wanner, George M Church

The large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by site-specific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

32: Single-Nucleotide-Resolution Computing and Memory in Living Cells
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Posted to bioRxiv 15 Feb 2018

Single-Nucleotide-Resolution Computing and Memory in Living Cells
2,319 downloads synthetic biology

Fahim Farzadfard, Nava Gharaei, Yasutomi Higashikuni, Giyoung Jung, Jicong Cao, Timothy K Lu

Computing and memory in living cells are central to encoding next-generation therapies and studying in situ biology, but existing strategies have limited encoding capacity and are challenging to scale. To overcome this bottleneck, we developed a highly scalable, robust and compact platform for encoding logic and memory operations in living bacterial and human cells. This platform, named DOMINO for DNA-based Ordered Memory and Iteration Network Operator, converts DNA in living cells into an addressable, readable, and writable computation and storage medium via a single-nucleotide resolution read-write head that enables dynamic and highly efficient DNA manipulation. We demonstrate that the order and combination of DNA writing events can be programmed by biological cues and multiple molecular recorders can be coordinated to encode a wide range of order-independent, sequential, and temporal logic and memory operations. Furthermore, we show that these operators can be used to perform both digital and analog computation, and record signaling dynamics and cellular states in a long-term, autonomous, and minimally disruptive fashion. Finally, we show that the platform can be functionalized with gene regulatory modules and interfaced with cellular circuits to continuously monitor cellular phenotypes and engineer gene circuits with artificial learning capacities. We envision that highly scalable, compact, and modular DOMINO operators will lay the foundation for building robust and sophisticated synthetic gene circuits for numerous biotechnological and biomedical applications.

33: Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs
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Posted to bioRxiv 15 May 2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs
2,275 downloads synthetic biology

Kale Kundert, James E Lucas, Kyle E Watters, Christof Fellmann, Andrew H Ng, Benjamin M Heineike, Christina M Fitzsimmons, Benjamin L Oakes, David F Savage, Hana El-Samad, Jennifer A Doudna, Tanja Kortemme

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

34: Loop Assembly: a simple and open system for recursive fabrication of DNA circuits
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Posted to bioRxiv 15 Jan 2018

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits
2,243 downloads synthetic biology

Bernardo Pollak, Ariel Cerda, Mihails Delmans, Simón Álamos, Tomás Moyano, Anthony West, Rodrigo A Gutiérrez, Nicola Patron, Fernán Federici, Jim Haseloff

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard Level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

35: A simple, robust, and low-cost method to produce the PURE cell-free system
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Posted to bioRxiv 18 Sep 2018

A simple, robust, and low-cost method to produce the PURE cell-free system
2,214 downloads synthetic biology

Barbora Lavickova, Sebastian J. Maerkl

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The OnePot method makes the PURE system easy to generate and allows it to be readily optimized and modified

36: Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly
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Posted to bioRxiv 25 May 2018

Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly
2,211 downloads synthetic biology

Wenqiang Li, Shuntang Li, Jie Qiao, Fei Wang, Yang Liu, Ruyi He, Yi Liu, Lixin Ma

CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

37: CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation
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Posted to bioRxiv 26 Feb 2017

CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation
2,198 downloads synthetic biology

Biliang Hu, Jiangtao Ren, Yanping Luo, Brian Keith, Regina M. Young, John Scholler, Yangbing Zhao, Carl H. June

Interleukin 18 (IL18) is known to induce the expression of interferon‐γ (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.

38: Complete absence of thebaine biosynthesis under home-brew fermentation conditions
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Posted to bioRxiv 13 Aug 2015

Complete absence of thebaine biosynthesis under home-brew fermentation conditions
2,154 downloads synthetic biology

Drew Endy, Stephanie Galanie, Christina D Smolke

Yeast-based biosynthesis of medicinal compounds traditionally derived from plant materials is improving. Both concerns and hopes exist for the possibility that individual small volume batch fermentations could provide distributed and independent access to a diversity of compounds some of which are now abused, illegal, or unavailable to many who need for genuine medical purposes. However, there are differences between industrial bioreactors and ‘home-brew’ fermentation. We used engineered yeast that make thebaine, a morphinan opiate, to quantify if differences in fermentation conditions impact biosynthesis yields. We used yeast that make an English ale as a positive fermentation control. We observed no production of thebaine and miniscule amounts of reticuline, an upstream biosynthetic intermediate, in home-brew fermentations; the positive control was palatable. We suggest that additional technical challenges, some of which are unknown and likely unrelated to optimized production in large-volume bioreactors, would need to be addressed for engineered yeast to ever realize home-brew biosynthesis of medicinal opiates at meaningful yields.

39: Recombineering in Vibrio natriegens
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Posted to bioRxiv 24 Apr 2017

Recombineering in Vibrio natriegens
2,129 downloads synthetic biology

Henry H Lee, Nili Ostrov, Michaela A Gold, George M Church

Here, we show that λ-Red homologs found in the Vibrio-associated SXT mobile element potentiate allelic exchange in V. natriegens by 10,000-fold. Specifically, we show SXT-Beta (s065), SXT-Exo (s066), and λ-Gam proteins are sufficient to enable recombination of single- and double- stranded DNA with episomal and genomic loci. We characterize and optimize episomal oligonucleotide-mediated recombineering and demonstrate recombineering at genomic loci. We further show targeted genomic deletion of the extracellular nuclease gene dns using a double-stranded DNA cassette. Continued development of this recombination technology will advance high-throughput and large-scale genetic engineering efforts to domesticate V. natriegens and to investigate its rapid growth rate.

40: Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae
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Posted to bioRxiv 21 Jul 2014

Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae
1,993 downloads synthetic biology

Owen W Ryan, Jeffrey M Skerker, Matthew J Maurer, Xin Li, Jordan C Tsai, Snigdha Poddar, Michael E Lee, Will DeLoache, John E Dueber, Adam P Arkin, Jamie H D Cate

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

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