scholarly journals Universal Loop assembly (uLoop): open, efficient, and species-agnostic DNA fabrication

2019 ◽  
Author(s):  
Bernardo Pollak ◽  
Tamara Matute ◽  
Isaac Nuñez ◽  
Ariel Cerda ◽  
Constanza Lopez ◽  
...  
Keyword(s):  
Expression Vectors ◽  
Assembly Systems ◽  
Dna Assembly ◽  
Golden Gate ◽  
Proof Of Concept ◽  
Biological Engineering ◽  
Dna Libraries ◽  
Dna Elements ◽  
Transcriptional Units ◽  
The Stability

ABSTRACTStandardised Type IIS DNA assembly methods are becoming essential for biological engineering and research. Although a ‘common syntax’ has been proposed to enable higher interoperability between DNA libraries, Golden Gate (GG)-based assembly systems remain specific to target organisms. Furthermore, these GG assembly systems become laborious and unnecessarily complicated beyond the assembly of 4 transcriptional units. Here, we describe “universal Loop” (uLoop) assembly, a simple system based on Loop assembly that enables hierarchical fabrication of large DNA constructs (> 30 kb) for any organism of choice. uLoop comprises two sets of four plasmids that are iteratively used as odd and even levels to compile DNA elements in an exponential manner (4n-1). The elements required for transformation/maintenance in target organisms are also assembled as standardised parts, enabling customisation of host-specific plasmids. Thus, this species-agnostic method decouples efficiency of assembly from the stability of vectors in the target organism. As a proof-of-concept, we show the engineering of multi-gene expression vectors in diatoms, yeast, plants and bacteria. These resources will become available through the OpenMTA for unrestricted sharing and open-access.Abstract Figure

scholarly journals Universal loop assembly: open, efficient and cross-kingdom DNA fabrication

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Bernardo Pollak ◽  
Tamara Matute ◽  
Isaac Nuñez ◽  
Ariel Cerda ◽  
Constanza Lopez ◽  
...  
Keyword(s):  
High Efficiency ◽  
Final Host ◽  
Expression Vectors ◽  
Assembly Systems ◽  
Dna Assembly ◽  
Golden Gate ◽  
Proof Of Concept ◽  
Biological Engineering ◽  
Dna Libraries ◽  
Multigene Expression

Abstract Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a ‘common syntax’ that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe ‘universal Loop’ (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.

scholarly journals Mobius Assembly: A Versatile Framework For Golden Gate Assembly

2017 ◽  
Author(s):  
Andreas I. Andreou ◽  
Naomi Nakayama
Keyword(s):  
Synthetic Biology ◽  
Drop Out ◽  
Dna Assembly ◽  
Dna Fragments ◽  
Golden Gate ◽  
Transcriptional Units ◽  
Combinatorial Assembly

Golden Gate Assembly is a powerful synthetic biology tool, which utilizes Type IIS enzymes for unidirectional assembly of multiple DNA fragments. The simplicity of its DNA assembly and the exchangeability of standard parts greatly facilitate the generation of combinatorial assembly libraries. Currently there are two popular Golden Gate Assembly frameworks that allow multigene augmentation (MoClo and Golden Braid); they render either high cloning capacity or vector toolkit simplicity. We have developed a new Golden Gate Assembly framework called Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. Mobius Assembly is based on a two-level approach and embraces the standard overhangs defined by MoClo and Golden Braid to confer exchangeability, but with reduced domestication requirement. Furthermore, we have implemented drop-out cassettes encoding chromogenic proteins for visible cloning screening. As proofs of concept, we have functionally assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Controlling Origami Stability Profile Using Magnets

2018 ◽  
Author(s):  
Hongbin Fang ◽  
Tse-Shao Chang ◽  
K. W. Wang
Keyword(s):  
Potential Energy ◽  
Wide Spectrum ◽  
Concept Design ◽  
Proof Of Concept ◽  
Potential Energy Landscape ◽  
Mechanical Metamaterials ◽  
Elastic Potential Energy ◽  
The Stability ◽  
Novel Applications ◽  
Stable Structures

Multi-stable structures and materials have attracted extensive research interests because they can provide a wide spectrum of adaptive properties and functionalities. Recently, origami has been identified as an important source for achieving multi-stability and has been exploited for developing unconventional mechanical metamaterials and metastructures. Once the crease pattern and the constituent materials have been specified for an origami structure, its multi-stability profile becomes unchangeable. On the other hand, a controllable profile would be desirable to endow the origami structures and origami metamaterials with further adaptability and versatility. This research investigates how to integrate magnets with origami to fundamentally alter the stability profiles. By embedding magnets into the origami facets or vertices, the magnetic potential energy would modify the original elastic potential energy landscape both quantitatively and qualitatively. Taking the stacked Miura-ori structures as examples, we show that different magnet assignments could either enrich the original bistable profile into a tri-stable or quad-stable profile, or simplify it into a mono-stable profile. Simultaneously, such magnet-induced evolutions of stability profile would trigger essential changes of the structure’s mechanical properties, which are promising to be used for developing multi-functional devices or metamaterials/metastructures. In this paper, in addition to the analyses, proof-of-concept design and prototype are presented. The results of this research would open up a new path for designing origami structures and metamaterials with controllable stability profiles that can be harnessed for many novel applications.

scholarly journals Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

2017 ◽  
Author(s):  
Vincent A Bielinski ◽  
Tayah M Bolt ◽  
Christopher L Dupont ◽  
Philip D Weyman
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Expression Vectors ◽  
Dna Assembly ◽  
Destination Vector ◽  
Genetic Tools ◽  
Entry Vector ◽  
Polymerase Iii ◽  
Recombination System ◽  
Low Efficiency ◽  
Gateway Entry Vector

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

scholarly journals pET expression vector customized for efficient seamless cloning

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 384-387
Author(s):  
Dragana Dobrijevic ◽  
Lily A Nematollahi ◽  
Helen C Hailes ◽  
John M Ward
Keyword(s):  
Expression Vector ◽  
Gene Replacement ◽  
Expression Vectors ◽  
Gram Negative Bacteria ◽  
Golden Gate ◽  
Gram Negative ◽  
Large Numbers ◽  
Parallel Cloning ◽  
Natural Enzyme

Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.

scholarly journals Hovering Stability of Helicopters With Elastic Constraints

2010 ◽  
Author(s):  
Paul E. I. Pounds ◽  
Aaron Dollar
Keyword(s):  
Proof Of Concept ◽  
Free Flight ◽  
Contact Constraint ◽  
Grasping And Manipulation ◽  
Reaction Forces ◽  
Manipulation Task ◽  
The Stability ◽  
External Forces ◽  
Elastic Constraints ◽  
Constraint Model

Aerial vehicles are difficult to stabilize, especially when acted upon by external forces. A hovering vehicle in contact with objects and surfaces must maintain flight stability while subject to forces imparted to the airframe through the point of contact. These forces couple with the motion of the aircraft to produce distinctly different dynamics from free flight. While external contact is generally avoided, extending aerial robot functionality to include contact with the environment during flight opens up new and useful areas such as perching, object grasping and manipulation. In this paper, we present a general elastic contact constraint model and analyze helicopter stability in the presence of those contacts. As an example, we evaluate the stability of a proof-of-concept helicopter system for manipulating objects using a compliant gripper that can be modeled as an elastic linkage with angular reaction forces. An off-the-shelf PID flight controller is used to stabilize the helicopter in free flight, as well as during the aerial manipulation task. We show that the planar dynamics of the object-helicopter system in vertical, horizontal and pitch motion around equilibrium are shown to remain stable, within a range of contact stiffnesses, under unmodified PID control.

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals Improving the Selectivity of an Osseointegrated Neural Interface: Proof of Concept For Housing Sieve Electrode Arrays in the Medullary Canal of Long Bones

2021 ◽  
Vol 15 ◽  
Author(s):  
Augusto X. T. Millevolte ◽  
Aaron M. Dingle ◽  
Jared P. Ness ◽  
Joseph Novello ◽  
Weifeng Zeng ◽  
...  
Keyword(s):  
Medullary Canal ◽  
Neural Interface ◽  
Proof Of Concept ◽  
Electrode Arrays ◽  
Prosthetic Control ◽  
Cuff Electrode ◽  
Cortical Responses ◽  
The Stability ◽  
And Function ◽  
Working Hypotheses

Sieve electrodes stand poised to deliver the selectivity required for driving advanced prosthetics but are considered inherently invasive and lack the stability required for a chronic solution. This proof of concept experiment investigates the potential for the housing and engagement of a sieve electrode within the medullary canal as part of an osseointegrated neural interface (ONI) for greater selectivity toward improving prosthetic control. The working hypotheses are that (A) the addition of a sieve interface to a cuff electrode housed within the medullary canal of the femur as part of an ONI would be capable of measuring efferent and afferent compound nerve action potentials (CNAPs) through a greater number of channels; (B) that signaling improves over time; and (C) that stimulation at this interface generates measurable cortical somatosensory evoked potentials through a greater number of channels. The modified ONI was tested in a rabbit (n = 1) amputation model over 12 weeks, comparing the sieve component to the cuff, and subsequently compared to historical data. Efferent CNAPs were successfully recorded from the sieve demonstrating physiological improvements in CNAPs between weeks 3 and 5, and somatosensory cortical responses recorded at 12 weeks postoperatively. This demonstrates that sieve electrodes can be housed and function within the medullary canal, demonstrated by improved nerve engagement and distinct cortical sensory feedback. This data presents the conceptual framework for housing more sophisticated sieve electrodes in bone as part of an ONI for improving selectivity with percutaneous connectivity toward improved prosthetic control.

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