scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

scholarly journals A Bacterial Expression Vector Archive(BEVA) for flexible modular assembly of Golden Gate-compatible vectors

2018 ◽  
Author(s):  
Barney A. Geddes ◽  
Marcela A. Mendoza-Suárez ◽  
Philip S. Poole
Keyword(s):  
Gene Expression ◽  
Host Range ◽  
Restriction Enzyme ◽  
Copy Number ◽  
Expression Vector ◽  
Assembly System ◽  
Broad Host Range ◽  
Golden Gate ◽  
Modular Assembly ◽  
Golden Gate Cloning

ABSTRACTWe 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-culturedRhizobium 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 byR. 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.IMPORTANCEModular 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 inEscherichia 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.

scholarly journals Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

2019 ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Synthetic Biology ◽  
Golden Gate ◽  
Flexible Design ◽  
Test Cycle ◽  
Design Build ◽  
Modular Cloning ◽  
Multiple Cloning Sites ◽  
Golden Gate Cloning

AbstractComplex multi-gene plasmids can be built from basic DNA parts in a reliable and automation friendly way using modular cloning standards, based on Golden Gate cloning. However, each toolkit or standard is limited to one or a few different vectors, which has led to an overabundance of toolkits with varying degrees of compatibility. Here, we present the Joint Universal Modular Plasmids (JUMP), a vector design that overcomes the limitations of current toolkits by expanding the paradigm of modular cloning: all vectors can be modified using modular cloning in an orthogonal way using multiple cloning sites. This allows researchers to introduce any feature into any JUMP vector and simplifies the Design-Build-Test cycle of synthetic biology. JUMP vectors are compatible with PhytoBrick basic parts, BioBricks and the Registry of Standard Biological Parts, and the Standard European Vector Architecture (SEVA). Due to their flexible design, JUMP vectors have the potential to be a universal platform for synthetic biology regardless of host and application. A collection of JUMP backbones and microbial PhytoBrick basic parts are available for distribution.

scholarly journals Rapid evolution at the Drosophila telomere: transposable element dynamics at an intrinsically unstable locus

Genetics ◽  
2020 ◽  
Vol 217 (2) ◽  
Author(s):  
Michael P McGurk ◽  
Anne-Marie Dion-Côté ◽  
Daniel A Barbash
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Insertion Site ◽  
Rapid Evolution ◽  
Interspecific Variation ◽  
High Rate ◽  
Evolutionary Strategy ◽  
Control Mechanisms ◽  
Genetic Conflict ◽  
Number Variation

AbstractDrosophila telomeres have been maintained by three families of active transposable elements (TEs), HeT-A, TAHRE, and TART, collectively referred to as HTTs, for tens of millions of years, which contrasts with an unusually high degree of HTT interspecific variation. While the impacts of conflict and domestication are often invoked to explain HTT variation, the telomeres are unstable structures such that neutral mutational processes and evolutionary tradeoffs may also drive HTT evolution. We leveraged population genomic data to analyze nearly 10,000 HTT insertions in 85  Drosophila melanogaster genomes and compared their variation to other more typical TE families. We observe that occasional large-scale copy number expansions of both HTTs and other TE families occur, highlighting that the HTTs are, like their feral cousins, typically repressed but primed to take over given the opportunity. However, large expansions of HTTs are not caused by the runaway activity of any particular HTT subfamilies or even associated with telomere-specific TE activity, as might be expected if HTTs are in strong genetic conflict with their hosts. Rather than conflict, we instead suggest that distinctive aspects of HTT copy number variation and sequence diversity largely reflect telomere instability, with HTT insertions being lost at much higher rates than other TEs elsewhere in the genome. We extend previous observations that telomere deletions occur at a high rate, and surprisingly discover that more than one-third do not appear to have been healed with an HTT insertion. We also report that some HTT families may be preferentially activated by the erosion of whole telomeres, implying the existence of HTT-specific host control mechanisms. We further suggest that the persistent telomere localization of HTTs may reflect a highly successful evolutionary strategy that trades away a stable insertion site in order to have reduced impact on the host genome. We propose that HTT evolution is driven by multiple processes, with niche specialization and telomere instability being previously underappreciated and likely predominant.

Comparison of historical anatomic landmarks vs. ultrasound guidance for the selection of a needle insertion site for jugular central venous access

Resuscitation ◽  
2012 ◽  
Vol 83 (4) ◽  
pp. e113-e114 ◽  
Author(s):  
Michel Galinski ◽  
Jean Catineau ◽  
Karim Tazarourte ◽  
Nicole Dardel ◽  
Philippe Bertrand ◽  
...  
Keyword(s):  
Ultrasound Guidance ◽  
Central Venous Access ◽  
Insertion Site ◽  
Venous Access ◽  
Needle Insertion ◽  
Central Venous ◽  
Anatomic Landmarks ◽  
Selection Of

scholarly journals A modular cloning toolkit for genome editing in plants

2019 ◽  
Author(s):  
Florian Hahn ◽  
Andrey Korolev ◽  
Laura Sanjurjo Loures ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Higher Order ◽  
Plant Science ◽  
Golden Gate ◽  
Coding Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Science Community ◽  
Modular Cloning ◽  
Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

Microfluidics and Beyond – Devices for Applications in Biotechnology -

2004 ◽  
Vol 820 ◽  
Author(s):  
Martina Daub ◽  
Rolf M. Kaack ◽  
Oliver Gutmann ◽  
Chris P. Steinert ◽  
Remigius Niekrawietz ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Life Science ◽  
Life Sciences ◽  
Microarray Technology ◽  
Parallel Operation ◽  
Modern Life ◽  
Liquid Handling ◽  
Microtiter Plates ◽  
Integration Density ◽  
Volume Range

AbstractFor the performance of certain analytical and diagnostic tasks in modern Life Science applications high throughput screening (HTS) methods are essential. Miniaturization, parallelization and automation allow to decrease consumption of expensive materials and lead to faster analyzing times. The miniaturization of total assay volumes by the use of microtiter plates as well as the microarray technology have revolutionized the field of biotechnology and Life Sciences. Neither printing of microarrays with droplet volumes of several picoliters, nor handling of precious enzymes in the upper nanoliter range can be accomplished with traditional liquid handling devices like air displacement pipettes. The development of novel low volume liquid handling devices, which are subject to current research, addresses the diverse requirements shifting steadily to lower volumes. Various novel non-contact dispensing methods in the nanoliter and picoliter range are presented and classified according to their working principles like air displacement and direct displacement methods (TopSpot®, NanoJetTM, Dispensing Well PlateTM). Properties of the various methods are compared in terms of flexibility, integration density, speed of operation, precision, addressable volume range and amenability to multi-parallel operation.

Marine Operation of Gas Turbine Engines and Waterjet Pumps for Small Passenger Vessels

1979 ◽  
Author(s):  
S. M. Kowleski ◽  
C. D. Harrington
Keyword(s):  
Gas Turbine ◽  
San Francisco ◽  
High Speed ◽  
San Francisco Bay ◽  
Turbine Engines ◽  
Gas Turbine Engines ◽  
Golden Gate ◽  
Operational Problem ◽  
Golden Gate Bridge ◽  
Selection Of

This paper describes the planning, developmental, equipment selection and operational problem phases of the high-speed ferry system presently being operated on San Francisco Bay by the Golden Gate Bridge, Highway and Transportation District. The reasons for the selection of the vessel propulsion package consisting of gas turbine engines and waterjet pumps are discussed in some detail. Most importantly, the paper covers the problems experienced to date with this equipment in continuous marine operation.

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