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1. Isolierung von E. coli Cotransformanten <ol><li> Bereiten elektrokompetenten Zellen des entsprechenden E. coli-Stamm transformiert werden. Transfer zum 1 ml LB-Medium (Trypton, Hefeextrakt, NaCl bei 10, 5 und 10 g / L, respectively) einer Einzelkolonie des Stammes der Wahl, und bei 37 ° C unter Schütteln Bedingungen von 180 Umdrehungen pro Minute. Verdünne die Vorkultur 1: 500 in 25 ml frischem LB-Medium und Inkubation der Kultur bei 37 ° C. </li><li> Bei log-Phase (0,6 OD) Zentrifugieren Sie die Zellsuspension bei 5.000 × g für 20 min), und das Pellet in 10%, v / v eiskaltes steriles Glycerin-Wasser in die Hälfte des ursprünglichen Kulturvolumens. Wiederholen Sie diesen Schritt 4 Mal, jedes Mal, wenn die Halbierung der Resuspensionsvolumen. Schließlich das Pellet in Glycerin / Wasser und teilen Sie die Zellsuspension in 50 ul Aliquots. Bewahren Sie die Aliquots bei -80 ° C bis zu 6 Monaten. </li><li> Lösen Sie das gewünschte Plasmid in sterilem Wasser mit 0,5 mM EDTA ergänzt. Thaw auf ice ein Aliquot elektrokompetenter Zellen und Mischen mit einer geeigneten Menge (2,5-5 ng) des Vektors. Abgeben der Mischung in eine 0,1 cm-Küvette für Elektroporation und gelten 1,8 kV. </li><li> Die elektroporierten Zellen unmittelbar Transfer in 1 ml SOC-Medium (LB-Medium mit 0,2% w / v Glucose, 10 mM MgCl 2, 2,5 mM KCl ergänzt), Inkubation für 1 h unter Schütteln und schließlich dann 100 ul Aliquots auf Petrischalen, LB-Agar mit dem entsprechenden Antibiotikum. Inkubieren Sie O / N bei 37 ° C. </li><li> Reinige die Transformanten durch Ausstreichen von Einzelkolonien auf Petrischalen. </li><li> Bereiten von elektrokompetenten Zellen der primären Transformanten und die Schritte 1.1 bis 1.5 mit weiteren Plasmide transformieren. </li><li> Bereiten Glycerin Aktien der Co-Transformanten. Übertragen einer einzelnen Kolonie in LB mit den geeigneten Antibiotika ergänzt, bei 37 ° C unter Schütteln und bei log-Phase (0,6 OD) Zentrifuge die Zellen Suspension bei 5000 g für 20 min. Resuspend des Pellets in LB-Medium, das Antibiotika, 15% v / v Glycerin. Dispense in Aliquots und Lagerung ist - 80 ° C. </li></ol> 2. Die Co-Expression von α und ε-Untereinheiten von E. coli DNA Polymerase III <ol><li> Übertragen Sie mit einer sterilen Schleife eine kleine Menge des entsprechenden Stammes Glycerolstammlösung (zB TOP10 / PGOOD-ε243 und TOP10 / PGOOD-ε243-θ / pBADα1160, siehe Abbildung 1) in eine Petrischale mit LB-Medium und Antibiotika. Lassen Sie die Zellsuspension trocken auf die Petrischale, und streifen die Zellen Tröpfchen. Bei 37 ° CO / N. </li><li> Transfer mit einem sterilen Zahnstocher eine einzelne Kolonie zu 1 ml LB-Medium Antibiotika. Inkubieren 8 h bei 37 ° C. Verdünnen Sie die Vorkultur 1: 500 in frischem Medium und Inkubation bei 30 ° C für 15 Stunden. </li><li> Fügen IPTG, Arabinose, oder Arabinose und IPTG, 1 mM jeweils. Inkubiere bei 30 ° C für 2,5 Std. Sammeln Sie die Zellen, und die Pellets lagern bei -20 ° C. </li><li> Tau-Pellets auf Eis und Resuspendieren in Lysepuffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 2,5 mM Dithiothreitol, 1 mM Phenylmethylsulfonylfluorid (PMSF), pH 8). Homogenisierung vorsichtig die Zellsuspension mit einem kalten Glas Töpfer. </li><li> Ultraschallbehandlung bei 15 W für 15 Sekunden, gefolgt von 15 Sekunden Kühlintervall (4 Zyklen). </li><li> Zentrifugieren der Gesamtproteinextrakte bei 10.000 × g für 20 min bei 4 ° C, um die lösliche Fraktion zu gewinnen. </li><li> Analyse durch SDS-PAGE (12,5% Acrylamid) Aliquots jeder löslichen Proteinextrakt. Zu diesem Zweck übertragen 20 ul jeder löslichen Proteinextrakt in Eppendorf-Röhrchen, enthaltend 60 &amp; mgr; l H 2 O und 20 &amp; mgr; l Ladepuffer (250 mM Tris-HCl pH 6,8, 10 mM β-Mercaptoethanol, 10% (w / v) SDS, 50% (v / v) Glycerin, 0,25% (w / v) Bromphenolblau) eingestellt und die Mischung 5 min. Last 18 ul jeder Probe und führen Sie die Elektrophorese bei 140 V für ca. 1,5 Stunden. </li></ol> 3. Co-Expression der α-Untereinheit des Deinococcus durans DNA Polymerase III und ε-Untereinheit von E. coli DNA Polymerase III <ol><li> Bereiten Sie eine Vorkultur des E. coli-Stamm TOP10 / pBAD-α Dr / PGOOD-ε243 in 1 ml LB-Ampicillin-Tetracyclin Medium und Inkubation 8 h bei 37 ° C. Gebrauchslösung: 1: 1000 in frischem Medium und Inkubation O / N bei 37 ° C. </li><li> Verdünne 1: 100 in 200 ml frischem Medium, Inkubation bei 37 ° C für 3 Stunden inkubiert, anschließend für 3 h mit Arabinose und IPTG, 1 mM jedes induzieren. Ernten Sie die Zellen, und speichern Sie das Pellet bei -20 ° C. </li><li> Das Pellet in 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF. Homogenisieren und stören die Zellen wie in 2.4 und 2.5 beschrieben. </li><li> Unmittelbar Zentrifuge die Zellen Lysat bei 10.000 × g für 20 min. Entsorgen Sie das Pellet. Den Überstand mit einem Büchner-Trichter mit 3 Lagen Filterpapier ausgestattet. Bewerben leichtem Vakuum. Um eine übermäßige Schaumbildung zu vermeiden, halten das Vakuum-Erlenmeyerkolben auf Eis während der Filtration. </li><li> Bestimmen der Proteinkonzentration nach Bradford 14. </li></ol> 4. Gelfiltrationschromatographie <ol><li> Äquilibrieren einem mit Wassermantel 16x70 (200) Gelfiltrationssäule mit 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8. Laden auf die Säule die löslichen Proteinextrakt. Für die optimale Auflösung, verwenden Sie ein 1 ml Probenschleife. Führen Sie die Chromatographie bei 0,6 ml / min. Halten Säulentemperatur bei 4 ° C im gesamten. </li><li> Sammeln 0,9 ml-Fraktionen und zu analysieren durch SDS-PAGE. Transfer 20 ul jeder relevanten Fraktion in Eppendorf-Röhrchen, enthaltend 60 &amp; mgr; l H 2 O und 20 &amp; mgr; l Ladepuffer (250 mM Tris-HCl pH 6,8, 10 mM β-Mercaptoethanol, 10% (w / v) SDS, 50% ( v / v) Glycerol, 0,25% (w / v) Bromphenolblau) eingestellt und die Mischung 5 min. Last 18 ul jeder Probe und führen Sie die Elektrophorese bei 140 V für ca. 1,5 Stunden. </li><li> Bestimmen die 3&#39;-5&#39;-Exonuclease und der DNA-Polymerase-Aktivität von jeder Fraktion in 96-well Mikrotiterplatten. Assay wird die Exonukleaseaktivität mit Thymidin-5&#39;-monophosphat-p-nitrophenylester (p NP-TMP) als das Substrat 15. Abzuschätzen DNA-Polymerase-Aktivität unter Verwendung des PPX (Pyrophosphatase, Purinnucleosidphosphorylase, Xanthinoxidase) enzymgekoppelten Assay 16 und 2- (4-Jodphenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazoliumchlorid (INT) als Elektronenakzeptor 16. </li></ol> 5. Mutationsanalyse von Populationen Co-Expression des Wildtyp-ε-Untereinheit der E. coli-DNA-Polymerase III und die mutagene εD12A Variant <ol><li> Übertragen einer Einzelkolonie von E. coli TOP10, die das pBAD-ε und die pGOOD1-εD12A 11 Vektoren zu 1 ml LB-Medium Antibiotika. Inkubieren Sie O / N bei 37 ° C. </li><li> Verdünnen Sie die Vorkultur 1: 250 in 3-Kolben mit frischem Medium (10 ml), fügen Sie die entsprechenden Induktoren (Arabinose, IPTG oder Arabinose und IPTG 1 mM jeweils) und bei 37 ° C8 h. Bereiten parallel nicht-induzierten Kulturen. </li><li> Sammeln Aliquots von 1 ml, dann verdünnt 1: 500 in neue Kolben mit frischem Medium (10 ml) ergänzt oder nicht mit den entsprechenden Induktoren. Inkubieren Sie O / N bei 37 ° C. </li><li> Wiederholen Sie die Schritte 5.2 und 5.3 und sammeln Aliquots von 1 ml. </li><li> Bestimmung der Anzahl der Generationen in jeder Kultur aufgetreten. Übertragen auf LB-Platten, 100 &amp; mgr; l geeignete Verdünnungen des Inokulums und der Kultur. Inkubieren Sie O / N bei 37 ° C. Die Kolonien auf den LB-Platten, und berechnet den Logarithmus der Anzahl der im Inokulum vorhanden ist (log I) und in der Kultur am Ende des Wachstums (log C) Zellen. Um die Anzahl der Generationen zu bestimmen, verwenden Sie die Formel: (log C - log I) /0.301. </li><li> Zentrifuge bei 5000 × g für 20 min, und die Zellen werden in 1 ml 50 mM Tris-HCl pH 7,6, 50 mM NaCl. Um die Zellen durchlässig, fügen Sie 2-3 Tropfen Chloroform und Vortex für 20 Sekunden. </li><li> Bestimmen Sie die β-Glukosidase Aktivität jedes Aliquot in einer 96-Well-Mikroplatte, unter Verwendung von p-Nitrophenyl-β-D-glucopyranosid (PNPGluc) als Substrat. Hinzufügen zu jeder Vertiefung 100 ul der permeabilisierten Zellen und 100 ul Substrat (16 mg / ml Stammlösung in H 2 O). Achten Sie auf Luftblasen in den Vertiefungen zu vermeiden. Die Absorption bei 420 nm unter Verwendung eines Mikroplatten-Lesegerät und den entsprechenden Filter. </li></ol>

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The authors declare that they have no competing financial interests.

Proteine ​​können sich nicht in Ordnung sein, mit Regionen, deren Tertiärstruktur ist nicht auf eine begrenzte Anzahl von Konformationen, 25 beschränkt. Diese ungeordnete Proteine ​​sind in der Regel anfälliger für Aggregation 25, und deren Isolierung und Charakterisierung könnte eine schwierige Aufgabe darstellen. Die ε-Untereinheit der E. coli-DNA-Polymerase III verfügt über zwei getrennte Bereiche 26,27, und zwar: i) die N-ter-Domain, mit dem 3&#39;-5&#39;-Ex...

Die Einführung der Überexpression Technologien erhöht entweder die biochemische Untersuchungen von niedriger Kopienzahl Enzyme und die industrielle Herstellung von pharmakologisch aktiven Proteinen (zB Insulin). Seit der Einführung dieser Technologien wurden erhebliche Fortschritte erzielt worden, um die Ausbeute und Qualität von rekombinanten Proteinen zu erhöhen. Darüber hinaus wurden 1,2 prokaryotischen und eukaryotischen 3 Überexpression Systeme über die Jahre entwickelt und bietet sinnvolle Alternativen zu dem &quot;Arbeitspferd&quot; der Proteinbiotechnologie, also Escherichia coli. Insbesondere die Verfügbarkeit von alternativen Plattformen E. coli führte zur Produktion von rekombinanten Peptiden oder Proteinen, die post-translationale Modifikationen. Es sollte jedoch erwähnt werden, dass E. coli stellt noch immer den Organismus der Wahl für die Produktion rekombinanter Proteine. Dies ist auf mehrere Faktoren, von denen die relevantesten sein kannberücksichtigt: i) die Verfügbarkeit von einer ganzen Reihe von Überexpression Systeme (Expressionsvektoren und Stämme) für E. coli 1,2; ii) die kurze Generationszeiten und hohe Biomasseerträge, der E. coli in einer Vielzahl von reichen und synthetische Medien; iii) die leichte Manipulation entweder auf biochemischer und genetischer Ebene dieses Mikroorganismus; iv) die Isolierung der Stämme, die die Produktion von toxischen Proteinen 4; v) die Konstruktion von Stämmen mit homogenen Induktion auf Bevölkerungsebene 5,6. Darüber hinaus wurde kürzlich gezeigt, dass Expressionssysteme für die Produktion in E. coli von posttranslational modifizierten Proteinen entwickelt werden können, und 2 aufgebaut. Gegenwärtig wird die Proteinexpression vor allem zur monomeren oder homo-oligomere Proteine, deren hypersynthesis kann mit einem einzigen Gen in ein geeignetes Plasmid kloniert geführt werden erhalten. Doch vor kurzem war die Aufmerksamkeitfür den Bau von E. bezahlt coli-Protein co-Expressionssysteme, gegen die Produktion, in vivo, der Hetero oligomeren Komplexen 2. Interessanterweise frühen Experimenten von Protein Co-Expression adressiert die Interspezies-Montage von großen und kleinen Untereinheiten der Cyanobakterien Ribulose-1,5-Bisphosphat-Carboxylase / Oxygenase 7,8, und die Assoziation von abgeschnitten und in voller Länge Formen von HIV-1- Reverse-Transkriptase-9. Diese bahnbrechenden Studien gezeigt, dass Protein Co-Expression stellt eine leistungsstarke Alternative zu herkömmlichen in vitro Rekonstitution. Zusätzlich Protein Co-Expression in E. coli wurde verwendet, um verschiedene Proteine ​​tragen post-translationale Modifikationen 2 zu produzieren, um Proteine, die unnatürliche Aminosäuren 2 zu erhalten, und die Ausbeute an überexprimiert Membranproteine ​​2 zu erhöhen. Ferner wird das Potential der Protein Coexpression als Werkzeug zum E. verleihen coli Wettbewerbnce in Proteinsekretion ist aktiv untersucht 2. Zwei Hauptstrategien von Protein Co-Expression in E. coli können verfolgt werden: i) die Verwendung eines einzigen Plasmid, um die verschiedenen Gene Gastgeber zu überexprimiert wird; ii) die Verwendung von mehreren Plasmiden in einzelnen Zellen zu co-exprimieren, die Zielproteine. Im ersten Fall müssen die Kriterien für die Auswahl von Plasmid nicht von denen der traditionellen einzelnen Proteinexpression Experimente unterscheiden, auch wenn insbesondere Plasmide, die Tandem-Promotor / Operator-Elemente wurden für die Co-Expression 10 aufgebaut. Dieser erste Ansatz ist daher ganz einfach. Es sollte jedoch erwähnt werden, dass die Verwendung von einem einzigen Plasmid zu co-exprimieren verschiedene Proteine ​​vor zwei Hauptschwierigkeiten: i) die Molekularmasse des Vektors mit der Anzahl von bereitgestellten Gene, wodurch die Anzahl der co-exprimierten Proteine; ii) wenn mehrere Gene unter der Kontrolle eines einzelnen Promotors kloniert, können Polarität Verklee die Expression der Gene, die distal von dem Promotor. Die Verwendung von Doppel- oder Mehrfach Plasmide in Einzel E. coli-Zellen hat, um die Kompatibilität der Vektoren der Wahl zu erreichen, daher Ohne Zwang auf die förderfähigen Kombinationen von Plasmiden. Diese zweite Co-Expression Strategie verfügt jedoch den Vorteil, das die Molekülmasse von Vektoren und begrenzt Polarität. Wir haben vor kurzem gebaut ein Protein Co-Expressionssystem entwickelt, um die Pendel von Genen zwischen den Co-Expressionsplasmide 11 zu erleichtern. Insbesondere konstruierten wir das PGOOD Vektoren Serie, die relevanten Merkmale davon sind: i) einem p15A-Replikationsursprung, um die Kompatibilität der PGOOD Plasmide mit den kommerziellen Vektoren, die den ColE1 Ursprung (zB den pBAD-Serie 12) bereitzustellen; ii) ein Tetracyclin-Resistenz-Kassette; iii) die Anwesenheit von lac abgeleitete regulatorische Elemente, dh der Promotor-Operator (O 1) Paar und das lacI </em&gt; q Gen. Unter Verwendung eines geeigneten pBAD-PGOOD Paar, waren wir in der Lage, den katalytischen Kern von E. überexprimieren coli-DNA-Polymerase III, die aus drei verschiedenen Untereinheiten zusammengesetzt ist, das heißt, α (der 5&#39;-3&#39;-Polymerase), ε (der 3&#39;-5&#39;-Exonuklease) und θ (Stabilisierungs ε) 13. Insbesondere haben wir gezeigt, dass die Co-Expression des αεθ Komplex war streng abhängig von der zusätzlich zu dem E. coli-Kulturmedium sowohl IPTG und Arabinose, Auslösung Expression von PGOOD und pBAD, bzw. (1A). In diesem Bericht zeigen wir, wie Protein Co-Expression effizient Schwierigkeiten der schlechten Löslichkeit von einem Proteinkomplex Untereinheit verbunden zu lösen. Darüber hinaus zeigen wir, wie in vivo Protein Ergänzung Tests durchgeführt werden, und wir endlich über die Verwendung von Protein Co-Expression zu stimmen Mutationsfrequenz in E. berichten colich a. Zu diesem Zweck haben wir PGOOD-pBAD Paare geeignet, relevante Beispiele für jede Fallstudie veranschaulichen.

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the multifaceted benefits of protein co-expression in escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

Die ε-Untereinheit der E. coli-DNA-Polymerase III aus 243 Aminosäuren und verfügt Schwerlöslichkeit 17,18, wenn die Stoffreste 187-243 deletiert 17. Allerdings haben wir früher gezeigt haben, dass 11 die Co-Expression von Volllängen-α, ε und θ-Untereinheiten ergibt lösliche DNA-Polymerase III katalytischen Kern (Abbildung 1). Insbesondere unter Verwendung des pBAD-PGOOD Co-Expressionssystem, zeigten wir, dass: i) die Überexpression von α und ε-Unter...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Die vielfältigen Vorteile von Protein Co-expression in<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K Aufrufe.  University of Bologna. Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells. 

Serie: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Forschung

Biologie

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening von Fungal Endoglucanaseaktivität in Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapping Bakterielle Functional Netzwerke und Pathways in<em&gt; Escherichia Coli</em&gt; Verwendung von Synthetic Genetic Arrays

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Rapid-Protokoll für die Herstellung von Elektrokompetente<em&gt; Escherichia coli</em&gt; Und<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Erkennung von Live-<em&gt; Escherichia coli</em&gt; O157: H7-Zellen durch PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Nicht-chromatographischen Reinigung von rekombinantem Elastin-ähnliche Polypeptide und deren Fusionen mit Peptiden und Proteinen aus<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Interzelluläre Gap Junction-Kommunikation: Eine funktionelle Biomarker zur Beurteilung Unerwünschte Auswirkungen von Schadstoffen und Toxinen und gesundheitlichen Vorteile von Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Methode zur Kennzeichnung Transkripte in einzelnen Escherichia coli Zellen für Einzelmolekül-Fluoreszenz In Situ Hybridisierung Experimente