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 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 Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome

2008 ◽  
Vol 74 (21) ◽  
pp. 6756-6765 ◽  
Author(s):  
Jodi L. Humann ◽  
Brenda K. Schroeder ◽  
Michael W. Mortimer ◽  
Brent L. House ◽  
Svetlana N. Yurgel ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Open Reading Frames ◽  
Vector System ◽  
Destination Vector ◽  
Entry Vector ◽  
Gateway Entry Vector ◽  
Sugar Kinase

ABSTRACT The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

scholarly journals On-chip multiplexed single-cell patterning and controllable intracellular delivery

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Zaizai Dong ◽  
Yanli Jiao ◽  
Bingteng Xie ◽  
Yongcun Hao ◽  
Pan Wang ◽  
...  
Keyword(s):  
Single Cell ◽  
Low Voltage ◽  
Transfection Efficiency ◽  
Cell Damage ◽  
Intracellular Delivery ◽  
Cell Manipulation ◽  
Expression Vectors ◽  
Vacuum System ◽  
Low Efficiency ◽  
On Chip

Abstract Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy.

scholarly journals Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

2022 ◽  
Author(s):  
James A Sawitzke ◽  
Nina C Costantino ◽  
Ellen Hutchinson ◽  
Lynn Thomason ◽  
Donald L Court
Keyword(s):  
Dna Assembly ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Engineering Technology ◽  
E Coli ◽  
Linear Dna ◽  
Recombination System ◽  
Genetic Engineering Technology

Assembly of intact, replicating plasmids from linear DNA fragments introduced into bacterial cells, i.e. in vivo cloning, is a facile genetic engineering technology that avoids many of the problems associated with standard in vitro cloning. Here we report characterization of various parameters of in vivo linear DNA assembly mediated by either the RecET recombination system or the bacteriophage λ Red recombination system. As previously observed, RecET is superior to Red for this reaction when the terminal homology is 50 bases. Deletion of the E. coli xonA gene, encoding Exonuclease I, a 3′→5′ single-strand DNA exonuclease, substantially improves the efficiency of in vivo linear DNA assembly for both systems. Deletion of ExoI function allowed robust RecET assembly of six DNA segments to create a functional plasmid. The linear DNAs are joined accurately with very few errors. This discovery provides a significant improvement to previously reported in vivo linear DNA assembly technologies.

scholarly journals Gateway Entry Vector Library of Wolbachia pipientis Candidate Effectors from Strain wMel

2018 ◽  
Vol 7 (1) ◽  
Author(s):  
Irene L. G. Newton ◽  
Kathy B. Sheehan
Keyword(s):  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Wolbachia Pipientis ◽  
Entry Vector ◽  
Intracellular Symbiont ◽  
Host Cytoplasm ◽  
Type Iv ◽  
Gateway Entry Vector ◽  
Bacterial Effectors

Wolbachia pipientis is an intracellular symbiont that modifies host biology using a type IV secretion system to inject bacterial effectors into the host cytoplasm. We utilized a bioinformatics approach to predict Wolbachia effectors and cloned the candidates into an entry vector, which can be utilized for subsequent analyses.

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 Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

2018 ◽  
Author(s):  
George M. Taylor ◽  
Paweł M. Mordaka ◽  
John T. Heap
Keyword(s):  
Metabolic Engineering ◽  
Design Space ◽  
Combinatorial Libraries ◽  
Dna Assembly ◽  
Destination Vector ◽  
E Coli ◽  
Assembly Method ◽  
Wide Range ◽  
Combinatorial Assembly ◽  
Sequence Boundaries

ABSTRACTDNA assembly allows individual DNA constructs or designed mixtures to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways inE. coliresulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.GRAPHICAL ABSTRACT

scholarly journals Yeast expression vectors using RNA polymerase III promoters

Gene ◽  
1994 ◽  
Vol 151 (1-2) ◽  
pp. 209-214 ◽  
Author(s):  
Paul D. Good ◽  
David R. Engelke
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Yeast Expression ◽  
Expression Vectors ◽  
Polymerase Iii

scholarly journals Stabilization of a Plasmid Coding for a Heterologous Antigen in Salmonella enterica Serotype Typhi Vaccine Strain CVD908-htrA by Using Site-Specific Recombination

2006 ◽  
Vol 74 (7) ◽  
pp. 4383-4386 ◽  
Author(s):  
Jonathan C. Stephens ◽  
Michael J. Darsley ◽  
Arthur K. Turner
Keyword(s):  
Salmonella Enterica ◽  
Gene Cassette ◽  
Expression Vectors ◽  
Virulence Plasmid ◽  
Multicopy Plasmid ◽  
Heterologous Antigen ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Recombination System ◽  
Heterologous Antigens

ABSTRACTA gene cassette incorporating thecrs-rsdsite-specific recombination system from theSalmonella entericasubsp.entericaserovar Dublin virulence plasmid improved the inheritance inS. entericaserotype Typhi strain CVD908-htrAof a multicopy plasmid expression vector. Use of this recombination cassette may improve expression of heterologous antigens from multicopy plasmid expression vectors in attenuated bacterial vaccine strains.

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