Diese Forschung wird unterstützt durch die niederländische Technologie Stiftung STW (12385), gehört die niederländische Organisation für wissenschaftliche Forschung (NWO), und die ist teilweise finanziert durch das Ministry of Economic Affairs (OTP Grant 12385, MS).  Wir danken für die Bereitstellung von pCH20, das Rückgrat der BIBAC-GW Vektoren Carol M. Hamilton (Cornell University, USA).

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+PCR+of+Intact+Bacteria+(Colony+PCR).">Direct PCR of Intact Bacteria (Colony PCR).</a> Curr Protoc Microbiol. , A.3D.1-A.3D.7 (2016).</li><li>Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mol+Biol.">Mol Biol.</a> Current Protocols in Molecular Biology. 1, (2003).</li><li>Edwards, K., Johnstone, C., Thompson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+preparation+of+plant+genomic+DNA+for+PCR+analysis.">A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.</a> Nucleic Acids Res. 19 (6), 1349 (1991).</li><li>Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cetyltrimethyl+ammonium+bromide+(CTAB)+DNA+miniprep+for+plant+DNA+isolation.">Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation.</a> Cold Spring Harb Protoc. (3), (2009).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. , (2012).</li><li>Sosa, J. 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Die Autoren erklären keine finanziellen Interessenkonflikte oder andere Interessenkonflikte.

Entscheidend für die Erzeugung von gentechnisch veränderten Pflanzen mit einzelnen, intakte Integrationen von ein Transgen ist die Wahl der binären Vektor verwendet. BIBAC Familie Vektoren wurden zur Sequenzen von Interessen, viele Pflanzen Arten23,24,25,26,27,28zu liefern. BIBAC Vektoren, einschließlich BIBAC-GW, Ausbeut...

Beim Generieren von transgenen Pflanzen ist in der Regel das Ziel, die integrierte transgene stabil ausgedrückt zu haben. Dies kann durch intakte Einzelkopie Integrationen von ein Transgen. Mehrfachintegrationen können zu erhöhten Ausdruck ein Transgen, sondern auch Gen-silencing führen. Der transgene Silencing ist wahrscheinlicher, wenn die eingefügte Sequenzen in Tandem- oder invertierte Wiederholungen1,2,3,4angeordnet sind. Binäre Vektoren dienen als Shuttles in Agrobakterium-vermittelten Umwandlung Experimente durchführen, um die Sequenzen von Interesse in Pflanzengenomen liefern. Die Zahl der Integrationen in ein pflanzliches Genom ist abhängig von der Kopienzahl des binären Vektor in Agrobacterium Tumefaciens5,6. Viele häufig verwendete binäre Vektoren sind hohe Kopie Vektoren, und daher ergeben eine hohe durchschnittliche Transgen Exemplarzahl: 3,3 bis 4,9 Kopien in Arabidopsis5.
Die Anzahl der T-DNA Integrationen kann mithilfe von binäre Vektoren, bei denen eine niedrige Kopienzahl in A. Tumefaciens, z. B. BIBAC7oder durch die Einführung einer T-DNS von A. Tumefaciens Chromosom5gesenkt werden. Die durchschnittliche Anzahl der Transgen-Integrationen in solchen Fällen liegt unter 25,8,9,10. Durch die Single Copy in A. Tumefaciens, und auch in Escherichia coli, BIBAC-Derivate erhalten und Konstrukte, die so groß wie 150 kb11liefern können.
GW-kompatible BIBAC Vektoren10,12 erlauben einfache Einführung von Genen von Interesse in den Vektor durch Gateway zu klonen. Die Verwendung von Gateway-Technologie vereinfacht das Klonen Verfahren, aber auch häufige Probleme im Zusammenhang mit großen niedrige Kopienzahl Vektoren13,14, wie eine geringe DNA-Ausbeute und eine begrenzte Auswahl an einzigartigen Beschränkung überwindet Standorte für das Klonen7,11. Die pBIBAC-GW-Derivate sind entweder Widerstand zu Glufosinat-Ammonium (pBIBAC-BAR-GW) oder DsRed Fluoreszenz in Samenhüllen (pBIBAC-RFP-GW) in Pflanzen (Abbildung 1)10,12zur Auswahl zur Verfügung. Für beide Vektoren ist eine Kanamycin-Resistenz-Gen als Selektionsmarker in Bakterien verwendet.
Kombinieren Sie die pBIBAC-GW-Vektoren: (1) einfache Design und Genmanipulation in E. Coli, und (2) intakt Single Copy Integrationen in Planta bei hohem Wirkungsgrad. Die pBIBAC-GW Vektoren Rendite durchschnittlich 1,7 Integrationen in Arabidopsis mit etwa der Hälfte der transgenen Pflanzen mit einer einzigen integrierten T-DNA10.
Stabile Expression von transgenen ist eine Voraussetzung für die meisten gentechnisch veränderten Pflanzen erzeugt. Stabile Transgene Ausdruck kann durch intakte, Single Copy Integrationen erreicht werden. Arbeiten mit transgenen Pflanzen tragen intakte, Single Copy Integrationen ist jedoch noch wichtiger, wenn zum Beispiel das Ziel, die Effizienz der Chromatin-basierte Prozesse wie Mutagenese, Rekombination, oder Reparatur und die Abhängigkeit von diesen zu studieren Prozesse der Chromatinstruktur an der Insertionsstelle und genomische Position. Für unser Interesse um die Abhängigkeit des Oligonukleotids gerichtet Mutagenese (ODM) auf den lokalen genomische Kontext zu untersuchen war eine Reihe von Reporter Linien mit intakten, Single Copy Integrationen von ein Reportergen Mutagenese erzeugte (Abbildung 2)10. Mit diesem Satz von Linien, zeigte sich, dass die ODM-Effizienz variiert zwischen transgenen Loci integriert genomische Standorten trotz der Transgene Ausdruck ziemlich ähnlich.

<table><tbody><tr><td>Kanamycin sulphate monohydrate</td><td>Duchefa</td><td>K0126</td><td></td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa</td><td>G0124</td><td></td></tr><tr><td>Rifampicin</td><td>Duchefa</td><td>R0146</td><td></td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma</td><td>T-3383</td><td></td></tr><tr><td>DB3.1 competent cells</td><td>Thermo Scientific - Invitrogen</td><td>11782-018</td><td>One Shot &lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant &lt;em&gt;E. coli&lt;/em&gt; strain can be used instead&amp;nbsp;&amp;nbsp;</td></tr><tr><td>DH10B competent cells</td><td>Thermo Scientific - Invitrogen</td><td>18290-015</td><td></td></tr><tr><td>Gateway LR clonase enzyme mix&amp;nbsp;</td><td>Thermo Scientific - Invitrogen</td><td>11791-019</td><td></td></tr><tr><td>tri-Sodium citrate dihydrate</td><td>Merck</td><td>106432</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>EDTA disodium dihydrate</td><td>Duchefa</td><td>E0511</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Scientific&amp;nbsp;</td><td>EO0491</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Honeywell Fluka</td><td>13423</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Merck</td><td>108346</td><td></td></tr><tr><td>Electroporation Cuvettes, 0.1 cm gap</td><td>Biorad</td><td>1652089</td><td></td></tr><tr><td>Electroporator Gene Pulser</td><td>BioRad</td><td></td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Calbiochem</td><td>442613</td><td></td></tr><tr><td>D(+)-Maltose monohydrate 90%</td><td>Acros Organics</td><td>32991</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100</td><td></td></tr><tr><td>Silwet L-77</td><td>Fisher Scientific</td><td>NC0138454</td><td></td></tr><tr><td>Murashige Skoog medium</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Glufosinate-ammonium (Basta)</td><td>Bayer</td><td>79391781</td><td></td></tr><tr><td>Restriction enzymes</td><td>NEB</td><td></td><td></td></tr><tr><td>Ethidium Bromide</td><td>Bio-Rad</td><td>1610433</td><td></td></tr><tr><td>Electrophoresis system</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106498</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100316</td><td></td></tr><tr><td>Blotting nylon membrane Hybond N+</td><td>Sigma Aldrich</td><td>15358</td><td>or GE Healthcare Life Sciences (RPN203B)</td></tr><tr><td>Whatman 3MM Chr blotting paper</td><td>GE Healthcare Life Sciences</td><td>3030-931</td><td></td></tr><tr><td>dNTP</td><td>Thermo Fisher</td><td>R0181</td><td></td></tr><tr><td>Acetylated BSA</td><td>Sigma-Aldrich</td><td>B2518</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Merck</td><td>805740</td><td></td></tr><tr><td>Sephadex G-50 Coarse</td><td>GE Healthcare Life Sciences</td><td>17004401</td><td>or Sephadex G-50 Medium (17004301)</td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate&amp;nbsp;</td><td>US Biological</td><td>S5010</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Sigma-Aldrich</td><td>D7656</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>Merck</td><td>106346</td><td></td></tr><tr><td>Storage Phosphor screen and casette</td><td>GE Healthcare Life Sciences</td><td>28-9564-74</td><td></td></tr><tr><td>Phosphor imager</td><td>GE Healthcare Life Sciences</td><td>Typhoon FLA 7000</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>Stratalinker 1800</td><td></td></tr><tr><td>cling film (Saran wrap)</td><td>Omnilabo</td><td>1090681</td><td></td></tr><tr><td>Agarose</td><td>Thermo Scientific - Invitrogen</td><td>16500</td><td></td></tr><tr><td>Boric acid</td><td>Merck</td><td>100165</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Blauw&amp;rsquo;; DNA ladder.</td><td>MRC Holland</td><td>MCT8070</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Rood&amp;rsquo;; DNA ladder</td><td>MRC Holland</td><td>MCT8080</td><td></td></tr><tr><td>Hexanucleotide Mix</td><td>Roche</td><td>11277081001</td><td></td></tr><tr><td>Large-Construct Kit</td><td>Qiagen</td><td>12462</td><td></td></tr><tr><td>Heat-sealable polyethylene tubing, clear</td><td>various providers</td><td></td><td>the width of the tubing should be wider than that of blotting membrane</td></tr><tr><td>Heat sealer</td><td></td><td></td><td></td></tr><tr><td>Membrane filter disk</td><td>Merck</td><td>VSWP02500</td><td></td></tr><tr><td>Magnesium chloride</td><td>Merck</td><td>105833</td><td></td></tr><tr><td>Hybridization mesh</td><td>GE Healthcare Life Sciences</td><td>RPN2519</td><td></td></tr></tbody></table>

generating transgenic plants with single-copy insertions using bibac-gw binary vector

Beim Generieren von transgenen Pflanzen ist in der Regel das Ziel, stabile Expression ein Transgen zu haben. Dies erfordert eine einheitliche, intakte Integration des Transgens, Multi-Kopie-Integrationen häufig Gen-silencing ausgesetzt sind. Der Gateway-kompatiblen binären Vektor basierend auf bakterielle künstliche Chromosomen (pBIBAC-GW), wie andere pBIBAC-Derivate, ermöglicht das Einfügen von Single Copy transgene mit hohem Wirkungsgrad. Als eine Verbesserung der ursprünglichen pBIBAC hat eine Gateway-Kassette in pBIBAC-GW, geklont worden, so dass die Sequenzen von Interesse jetzt leicht in den Vektor Transfer DNA (T-DNA) durch Klonen Gateway integriert werden können. Die Transformation mit pBIBAC-GW wird häufig, einen Wirkungsgrad von 0,2 – 0,5 %, wobei die Hälfte der gentechnisch veränderten Pflanzen eine intakte Single Copy-Integration der T-DNA tragen. Die pBIBAC-GW-Vektoren sind mit Resistenz gegenüber Glufosinat-Ammonium oder DsRed Fluoreszenz in Samenhüllen in Pflanzen zur Auswahl und mit einer Resistenz gegen Kanamycin als Auswahl in Bakterien. Hier ist eine Reihe von Protokollen, die der Leser durch den Prozess der Erzeugung transgener Pflanzen mit pBIBAC-GW präsentiert: ausgehend von Rekombination die Sequenzen von Interesse in der pBIBAC-GW-Vektor der Wahl, um die Transformation mit Pflanzen Agrobacterium, Auswahl der gentechnisch veränderten Pflanzen und Versuchsanlagen für Unversehrtheit und Kopie Zahl der Einsätze mit DNA zu beflecken. Aufmerksamkeit gilt beim Entwerfen einer DNA-befleckenden Strategie, Single und copy Integrationen auf einzelne und mehrere Loci zu erkennen.

Mit dem BIBAC-GW-System waren Reporter Konstrukte für ein Studium ODM in Pflanzen erzeugten10. Konstrukte wurden in der Gateway-Eintrag Vektor pENTR-gm12 entworfen und in pBIBAC-BAR-GW (Abbildung 1) mit dem Gateway LR Rekombination Reaktion eingefügt.
Arabidopsis verwandelten sich mit pDM19, ein BIBAC-BAR-GW-Plasmid mit einer mTurquoise-eYFP Reporter...

Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Mit einem pBIBAC-GW binären Vektor macht Erzeugung transgene Pflanzen mit intakten Single Copy Einfügungen, ein einfacher Vorgang. Hier präsentiert eine Reihe von Protokollen, die führen des Lesers durch den Prozess der Generierung von transgenen Pflanzen in Arabidopsis und Versuchsanlagen für Unversehrtheit und Anzahl der Einsätze zu kopieren.

Generierung von transgenen Pflanzen mit Single-Copy-Einschübe mit binären Vektor BIBAC-GW

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

generating-transgenic-plants-with-single-copy-insertions-using-bibac

Research

JoVE Journal

Genetics

12.4K Aufrufe.  University of Amsterdam. Mit einem pBIBAC-GW binären Vektor macht Erzeugung transgene Pflanzen mit intakten Single Copy Einfügungen, ein einfacher Vorgang. Hier präsentiert eine Reihe von Protokollen, die führen des Lesers durch den Prozess der Generierung von transgenen Pflanzen in Arabidopsis und Versuchsanlagen für Unversehrtheit und Anzahl der Einsätze zu kopieren.

Serie: Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Transient Gene Expression in Tabak mit Gibson Versammlung und die Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Forschung

Umwelt

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

Pflanzliche Proteine in Insektenzellen abgesondert ändern Baculovirus Expressionsvektoren herstellen

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Biochemie

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Induzierbaren, Zelle Typ-spezifischen Ausdruck in Arabidopsis Thaliana durch LhGR-vermittelten Trans-Aktivierung

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Genetik

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Biotechnik

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Die MultiBac Protein Complex Produktion Platform am EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Biologie

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration und PVX Agroinfektion in Kartoffel und Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

<meta charset="utf8"></head><body>CRISPR-basierte effiziente Genom-Editierung für resistente Stärke in der Reiszüchtung

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Rationalisierung der Reiszüchtung mit CRISPR/Cas zur Erzielung optimaler phänotypischer und agronomischer Merkmale

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

Breeding by Design für funktionellen Reis mit Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip Transformation von Flachs (<em&gt; Linum usitatissimum</em&gt;), Um Transgene Progenies mit hoher Umwandlungsrate generieren

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Generierung von transgenen Pflanzen mit Single-Copy-Einschübe mit binären Vektor BIBAC-GW

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Transient Gene Expression in Tabak mit Gibson Versammlung und die Gene Gun

Pflanzliche Proteine in Insektenzellen abgesondert ändern Baculovirus Expressionsvektoren herstellen

Induzierbaren, Zelle Typ-spezifischen Ausdruck in Arabidopsis Thaliana durch LhGR-vermittelten Trans-Aktivierung

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

Die MultiBac Protein Complex Produktion Platform am EMBL

Agroinfiltration und PVX Agroinfektion in Kartoffel und Nicotiana benthamiana

Breeding by Design für funktionellen Reis mit Genome Editing Technologies

Breeding by Design für funktionellen Reis mit Genome Editing Technologies

Breeding by Design für funktionellen Reis mit Genome Editing Technologies

Floral-Dip Transformation von Flachs (