Ce travail a été soutenu par K08 Prix CA116429-04 de MC et par une subvention du cancer Conquer maintenant (CONCERN) Fondation # 8501

<ol>
	<li>Camps, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ColE1-like+plasmid+replication+for+recombinant+gene+expression.">Modulation of ColE1-like plasmid replication for recombinant gene expression.</a> Recent Pat DNA Gene Seq. 4, 58-73 (2010).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+replication+of+plasmid+ColE1+DNA+by+RNA+polymerase,+ribonuclease+H,+and+DNA+polymerase+I.">Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I.</a> Cold Spring Harb Symp Quant Biol. 43 Pt 1, 409-417 (1979).</li><li>Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ColE1+replication+control+circuitry:+sense+from+antisense.">ColE1 replication control circuitry: sense from antisense.</a> Cell. 55, 929-932 (1988).</li><li>Camps, M., Naukkarinen, J., Johnson, B. P., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+evolution+in+Escherichia+coli+using+a+highly+error-prone+DNA+polymerase+I.">Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.</a> Proc Natl Acad Sci U S A. 100, 9727-9732 (2003).</li><li>Shinkai, A., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mutagenesis+by+Escherichia+coli+DNA+polymerase+I.+Ile(709)+in+motif+A+functions+in+base+selection.">In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.</a> J Biol Chem. 276, 46759-46764 (2001).</li><li>Uyemura, D., Lehman, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+mutant+forms+of+DNA+polymerase+I+from+Escherichia+coli.+I.+The+polA12+mutation.">Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.</a> J Biol Chem. 251, 4078-4084 (1976).</li><li>Sedgwick, B., Robins, P., Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+removal+of+alkylation+damage+from+DNA+by+AlkB+and+related+DNA+dioxygenases.">Direct removal of alkylation damage from DNA by AlkB and related DNA dioxygenases.</a> Methods Enzymol. 408, 108-120 (2006).</li><li>Aharoni, A., Gaidukov, L., Khersonsky, O., Mc, Q. G. S., Roodveldt, C., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'evolvability'+of+promiscuous+protein+functions.">The 'evolvability' of promiscuous protein functions.</a> Nat Genet. 37, 73-76 (2005).</li><li>Elena, S. F., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+experiments+with+microorganisms:+the+dynamics+and+genetic+bases+of+adaptation.">Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation.</a> Nat Rev Genet. 4, 457-469 (2003).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoFormation+of+an+RNA+primer+for+initiation+of+replication+of+ColE1+DNA+by+ribonuclease+H.">FoFormation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.</a> Proc Natl Acad Sci U S A. 77, 2450-2454 (1980).</li><li>Bryan, S. K., Moses, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sufficiency+of+the+Klenow+fragment+for+survival+of+polC(Ts)+pcbA1+Escherichia+coli+at+43+degrees.">Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees.</a> C. J Bacteriol. 170, 456-458 (1988).</li></ol>

Cet article présente un protocole de mutagenèse qui permet la génération aléatoire des grandes banques de mutants sans la nécessité pour le clonage ou la PCR. Cette méthode est basée sur erreurs de réplication d&#39;un plasmide codant pour une séquence d&#39;intérêt. En théorie, les mutations devraient être largement limités aux 100-300 pb située immédiatement en aval de l&#39;interrupteur de l&#39;ARN / ADN, la taille de leader brin intermédiaire produite par Pol I 2,10. Nous avons constaté que dans les conditions présentées dans le présent protocole, Pol mutations se produisent partout dans I le plasmide, bien que diminuant la fréquence comme la distance entre les augmentations Ori plasmide (figure 3). Cette constatation implique que la transition ou &quot;switch&quot; de Pol I Pol III pendant ColE1 réplication plasmidique est beaucoup plus progressif que celui rapporté antérieurement, au moins dans nos conditions expérimentales 2, et est d&#39;accord avec les études antérieures suggérant une redondance fonctionnelle entre Pol I et Pol III 11. Notre protocole original décrit mutagenèse dans des cultures liquides (4). Ce protocole donne 0,41 mutations / Ko pour d &lt;1000, c&#39;est à dire au sein du BP 1000 adjacent à l&#39;ADN / ARN interrupteur (tableau 1). Hypersaturation en laissant le liquide de culture dans le shaker pendant 3 jours sans l&#39;ajout de toute nouvelle presse soulève la fréquence de mutation à 0,70 mutations / ko mais les résultats de rendement plasmidique très pauvres (moins de 1% par rapport au jour 1; données non présentées) ( Tableau 1). Nous présentons ici un protocole simplifié basé sur la transformation directe plaquage de la cible plasmide sur gélose solide à 37 ° C (figure 1). Cette procédure produit la plus grande fréquence de mutation / cycle de mutagenèse (0,92 mutations / Ko pour d &lt;1000) et facilite grandement l&#39;itération. L&#39;évolution des conditions de culture d&#39;un milieu solide affecte également le spectre de mutation (tableau 2). Mutagenèse ensemencement direct réduit le asymétrie marquée entre les substitutions de paires de bases complémentaires vu en culture liquide (comparer C → T vs G → A et A → G → T vs C). D&#39;autre part, l&#39;ensemencement direct a produit plus indels (de moins de 0,5% à 4%) et diminué le nombre de mutations G → A par 3 fois, conduisant à une surreprésentation de G / C mutations (74%). Globalement, nous pensons que la plus grande simplicité et une efficacité accrue du protocole ensemencement direct présentés ici l&#39;emporte sur les avantages de la gamme un peu plus équilibrée mutation produite par mutagenèse liquide. Dans le plasmide cible, les mutations générées par notre méthode ne sont pas limités à la séquence cible souhaitée. Cependant, l&#39;évolution du roman activités biochimiques devrait dépendre de mutations dans le gène cible, car elle représente une approche qualitative plutôt que d&#39;un changement quantitatif. Ainsi, en combinant nos mutagenèse plasmide avec le dépistage de mutants sur des plaques de gradient capitalise sur les atouts de notre système (les grandes bibliothèques et de la disponibilité de la sélection) pour l&#39;évolution des nouvelles propriétés biochimiques. D&#39;autres applications de mutagenèse aléatoire telles que l&#39;optimisation des activités enzymatiques existants ou randomisant des domaines spécifiques d&#39;un gène ne nécessitent clonage suivantes mutagenèse aléatoire. Dans ce cas, disposer de la bibliothèque dans un plasmide, par opposition à un produit d&#39;amplification PCR améliore l&#39;efficacité du clonage, en facilitant l&#39;amplification et la restriction. En somme, nous démontrons un protocole simple de créer une bibliothèque aléatoire mutantes pour un gène cible donné qui se distingue par sa simplicité et de la diversité des bibliothèques générés. Nous montrons comment cette méthode peut être couplée avec des sélections fonctionnelles pour l&#39;évolution efficace de nouvelles activités biochimiques. En outre, notre in vivo généré les bibliothèques peuvent être facilement clonées, permettant la mutagenèse dirigée ou optimisation des activités existantes.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>carbenicillin</td>
<td>&nbsp;</td>
<td>CellGro</td>
<td>46100R6</td>
<td>0.1mg/ml final</td>
</tr>
<tr>
<td>tetracycline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP7640</td>
<td>0.05mg/ml final</td>
</tr>
<tr>
<td>chloramphenicol</td>
<td>&nbsp;</td>
<td>Genlantis</td>
<td>M120100</td>
<td>0.03mg/ml final</td>
</tr>
<tr>
<td>LB Agar Miller</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP1425</td>
<td>&nbsp;</td>
<tr>
<td>LB Broth Miller</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244620</td>
<td>&nbsp;</td>
<tr>
<td>Soft Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>214580</td>
<td>Made in house</td>
</tr>
<tr>
<td>Acridine Orange</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A38401-1</td>
<td>&nbsp;</td>
<tr>
<td>Antifoam B emulsion</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5757</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>332030025</td>
<td>&nbsp;</td>
<tr>
<td>100x100x15mm sq dish</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>0875711A</td>
<td>&nbsp;</td>
<tr>
<td>100mm rnd dish </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>0875712A</td>
<td>&nbsp;</td>
<tr>
<td>Microscope slide 25x75x1mm</td>
<td>&nbsp;</td>
<td>Gold Seal</td>
<td>3048</td>
<td>&nbsp;</td>
<tr>
<td>Electroporator 2510</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Electroporator 2510</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2mm gap tubes</td>
<td>&nbsp;</td>
<td>MolBioproducts</td>
<td>5520</td>
<td>&nbsp;</td>
<tr>
<td>Zippy mini prep kit</td>
<td>&nbsp;</td>
<td>Zymo </td>
<td>D4020</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mutagenesis and functional selection protocols for directed evolution of proteins in e. coli

La génération efficace de la diversité génétique représente un outil précieux moléculaire qui peut être utilisé pour étiqueter synthèse de l&#39;ADN, afin de créer des signatures moléculaires uniques, ou de faire évoluer les protéines dans le laboratoire. Ici, nous présentons un protocole qui permet la génération de grande taille (&gt; 10 11) banques de mutants pour une séquence cible donnée. Cette méthode est basée sur la réplication d&#39;un plasmide d&#39;encodage ColE1 la séquence désirée par une variante basse-fidélité de l&#39;ADN polymérase I (Pol LF-I). Le plasmide cible est transformé en une souche de E. mutator coli et étalées sur un milieu solide, ce qui donne entre 0,2 et 1 mutations / ko, en fonction de la localisation du gène cible. Fréquences de mutation plus élevés sont obtenus par itération de ce processus de mutagenèse. Comparé à d&#39;autres méthodes de mutagenèse, notre protocole se distingue par sa simplicité, comme le clonage ou la PCR ne sont impliqués. Ainsi, notre méthode est idéale pour l&#39;étiquetage mutationnel de plasmides ou autres modèles, je Pol ou à explorer une grande partie de l&#39;espace de séquence pour l&#39;évolution des activités non présentes dans la cible originale. Le contrôle serré spatiale que la PCR ou randomisé à base d&#39;oligonucléotides offrent des méthodes peuvent aussi être atteints par clonage ultérieur des sections spécifiques de la bibliothèque. Ici nous fournir des protocoles montrant comment créer une bibliothèque aléatoire de mutants et comment établir des médicaments à base des sélections dans E. coli afin d&#39;identifier des mutants présentant de nouvelles activités biochimiques.

Watch this Scientific Journal Video about Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli at JoVE.com

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Ici nous démontrons un protocole simple de créer une bibliothèque aléatoire mutant pour une séquence cible donnée. Nous montrons comment cette méthode, qui est réalisée in vivo chez Escherichia coli, peuvent être couplées avec des sélections fonctionnelles d&#39;évoluer de nouvelles activités enzymatiques.

Protocoles de sélection mutagenèse et fonctionnelles pour l&#39;évolution dirigée de protéines dans les E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

mutagenesis-functional-selection-protocols-for-directed-evolution

Research

JoVE Journal

Biology

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

30.2K vues.  University of California Santa Cruz - UCSC. Ici nous démontrons un protocole simple de créer une bibliothèque aléatoire mutant pour une séquence cible donnée. Nous montrons comment cette méthode, qui est réalisée in vivo chez Escherichia coli, peuvent être couplées avec des sélections fonctionnelles d'évoluer de nouvelles activités enzymatiques.

Vidéo: Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation d&#39;ADN plasmidique dans E. coli en utilisant la méthode du choc thermique

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

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Biologie

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagenèse dirigée maison des plasmides entiers

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristallisant protéines membranaires pour déterminer la structure à l&#39;aide mésophases lipidique

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

L&#39;examen de la dynamique conformationnelle des protéines membranaires In situ Avec site-dirigé marquage fluorescent

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Utilisation d&#39;un robot pour haut-débit cristallisation des protéines membranaires dans des mésophases lipidiques

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protocoles pour la mise en œuvre d&#39;un<em&gt; Escherichia coli</em&gt; Sur la base du système d&#39;expression cellulaire gratuit TX-TL pour la biologie synthétique

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expression quantitative et purification appliquée à recombinant disulfure riches protéines venin produit dans<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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 base Expression dépistage des protéines membranaires dans<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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.