Ringraziamo Jongmin Kim, Dan Siegal-Gaskins, Anu Thubagere, e Enoch Yeung per l&#39;assistenza razionalizzare il protocollo, e Chiara Chen e Barclay Lee per l&#39;assistenza nelle prime fasi del progetto. Questo materiale si basa su lavoro sostenuto in parte dalla Defense Advanced Research Projects Agency (DARPA / OMT) del programma Living Fonderie, numero di contratto HR0011-12-C-0065 (DARPA / CMO.ZZS è supportato anche da un UCLA / Caltech Medical Scientist Fellowship Program Formazione e da un DoD, Air Force Office of Scientific Research, Science National Defense and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168. Le opinioni e le conclusioni contenute in questo documento sono quelle degli autori e non deve essere interpretato come rappresentazione ufficialmente politiche, esplicitamente o implicita, della Defense Advanced Research Projects Agency o il governo degli Stati Uniti.

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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> Transcription and Translation: A Practical Approach. , 179-209 (1984).</li><li>Kim, H. C., Kim, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+energizing+cell-free+protein+synthesis.">Methods for energizing cell-free protein synthesis.</a> Journal of bioscience and bioengineering. 108, 1-4 (2009).</li><li>Michel-Reydellet, N., Calhoun, K., Swartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+stabilization+for+cell-free+protein+synthesis+by+modification+of+the+Escherichia+coli+genome.">Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome.</a> Metabolic engineering. 6, 197-203 (2004).</li><li>Liu, D. V., Zawada, J. F., Swartz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streamlining+Escherichia+coli+S30+extract+preparation+for+economical+cell-free+protein+synthesis.">Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis.</a> Biotechnology progress. 21, 460-465 (2005).</li><li>Andrianantoandro, E., Basu, S., Karig, D. K., Weiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology:+new+engineering+rules+for+an+emerging+discipline.">Synthetic biology: new engineering rules for an emerging discipline.</a> Molecular systems biology. 2, 2006.0028 (2006).</li><li>Kwok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+hard+truths+for+synthetic+biology.">Five hard truths for synthetic biology.</a> Nature. 463, 288-290 (2010).</li><li>Tabor, S., Richardson, C. 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+bacteriophage+T7+RNA+polymerase/promoter+system+for+controlled+exclusive+expression+of+specific+genes.">A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 1074-1078 (1985).</li><li>Lewicki, B. T., Margus, T., Remme, J., Nierhaus, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+rRNA+transcription+and+ribosomal+assembly+in+vivo.+Formation+of+active+ribosomal+subunits+in+Escherichia+coli+requires+transcription+of+rRNA+genes+by+host+RNA+polymerase+which+cannot+be+replaced+by+bacteriophage+T7+RNA+polymerase.">Coupling of rRNA transcription and ribosomal assembly in vivo. Formation of active ribosomal subunits in Escherichia coli requires transcription of rRNA genes by host RNA polymerase which cannot be replaced by bacteriophage T7 RNA polymerase.</a> Journal of molecular biology. 231, 581-593 (1993).</li><li>Iskakova, M. B., Szaflarski, W., Dreyfus, M., Remme, J., Nierhaus, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+coupled+in+vitro+transcription-translation+system+derived+from+Escherichia+coli+cells:+synthesis+of+high-yield+fully+active+proteins.">Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins.</a> Nucleic acids research. 34, e135 (2006).</li><li>Kigawa, T., 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=Preparation+of+Escherichia+coli+cell+extract+for+highly+productive+cell-free+protein+expression.">Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.</a> Journal of structural and. 5, 63-68 (2004).</li><li>Matsuda, T., 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=Improving+cell-free+protein+synthesis+for+stable-isotope+labeling.">Improving cell-free protein synthesis for stable-isotope labeling.</a> Journal of biomolecular. NMR. 37, 225-229 (2007).</li><li>Sitaraman, K., 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=A+novel+cell-free+protein+synthesis+system.">A novel cell-free protein synthesis system.</a> Journal of biotechnology. 110, 257-263 (2004).</li><li>Becskei, A., Serrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+stability+in+gene+networks+by+autoregulation.">Engineering stability in gene networks by autoregulation.</a> Nature. 405, 590-593 (2000).</li><li>Maeda, H., Fujita, N., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+among+seven+Escherichia+coli+sigma+subunits:+relative+binding+affinities+to+the+core+RNA+polymerase.">Competition among seven Escherichia coli sigma subunits: relative binding affinities to the core RNA polymerase.</a> Nucleic acids research. 28, 3497-3503 (2000).</li><li>Caschera, F., 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=Coping+with+complexity:+machine+learning+optimization+of+cell-free+protein+synthesis.">Coping with complexity: machine learning optimization of cell-free protein synthesis.</a> Biotechnology and bioengineering. 108, 2218-2228 (2011).</li><li>Hodgman, C. 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Gli autori dichiarano di non avere interessi finanziari in competizione.

Sistema di espressione cell-free TX-TL basata Il Escherichia coli endogena qui descritto è una reazione di facile conduzione tre tubo che può richiedere meno di otto ore da impostare alla raccolta dei dati. Il processo di creazione di tutti i reagenti richiede tempo totale di cinque giorni (con notevoli esigenze di lavoro in un solo giorno), ma produce estratto grezzo di 3.000 reazioni e buffer-making reagenti per 10.000 reazioni (Figura 1). Inoltre, estratto grezzo e buffer facendo reagenti sono stabili per almeno 1 anno a -80 ° C, consentendo molteplici usi di una preparazione di 4. A $ 0,11 per 10 reazione microlitri (0,26 dollari compreso il lavoro), i costi sono inferiori del 98% rispetto comparabile sistemi commerciali (Figura 2). Ci sono alcune limitazioni irrisolti, tuttavia, al sistema. L&#39;efficienza fine di ciascun preparato estratto cellulare grezzo può variare sulla base di competenza degli utenti e delle condizioni ambientali, sebbene tvariazione di rendimento ypical è compresa tra il 5-10% (Fig. 4b). Come risultato, lotto a lotto variabilità sia espressione end-point e nelle dinamiche di espressione sono da aspettarsi. Queste variazioni saranno probabilmente rimarrà fino estratto è completamente caratterizzata o fino a quando la creazione estratto è completamente automatizzato. Se il sistema di libera espressione-cella viene utilizzata per condurre esperimenti quantitativi sensibili, si consiglia di eseguire tutti gli esperimenti con la stessa produzione di estratto cellulare grezzo. La resa da un unico lotto estratto cellulare greggio, circa 3000 reazioni, dovrebbe essere sufficiente per i corsi sperimentali tipici. Anche se abbiamo il sospetto variazione può essere rimosso da scalare e automatizzare la procedura, tali tentativi comporterebbe un investimento di risorse notevole. Inoltre, anche se i livelli di espressione end-point sono ragionevolmente facile da determinare, più lavoro deve essere fatto in comprensione dinamica intrinseca al sistema cell-free. È noto che sia CONCORRENZ risorsan risorse e limitazione può influenzare le dinamiche di espressione. Ad esempio, limitato sigma endogeno 70 può risultare in un regime saturare con maggiore DNA stampo produrre un profilo di espressione analoga a quella del nucleotide o amminoacido esaurimento. 9,27 Tuttavia, dinamiche non devono essere pienamente compreso per utilizzare il sistema. Per gli aumenti puri di rendimento, ottimizzazione può essere fatto da approcci di machine-learning. 28 Questioni di competizione per le risorse e di limitazione possono essere affrontate da modelli matematici verificata utilizzando dati sperimentali. Il protocollo qui presentato è ottimizzato per un ceppo BL21-Rosetta2, ma è generalizzabile ad altri E. coli. Modifiche in BL21-Rosetta2, quali la rimozione del gene codificante lon proteasi e l&#39;aggiunta di geni codificanti tRNA rari, permettono la produzione di proteine ​​massima. Abbiamo tentato il protocollo con altri due ceppi di estratto - BL21 solo e BL21 trxA ko-and ha trovato il 50% in meno di rendimento di proteine. Noi ipotizziamo che i rendimenti allo stesso modo diminuiscono quando si utilizzano altri ceppi. Altre variazioni di parametri, come il passaggio terreno di coltura 2xYT per LB ed altri brodi ricchi, hanno portato a una diminuzione dei rendimenti delle proteine. Sistemi di espressione cell-free che utilizzano i macchinari di trascrizione-traduzione endogena ed esogena e meccanismi di regolazione hanno ampie applicazioni in proteine ​​e metaboliti di espressione e di biologia sintetica. 3,29 Invece di essere limitato a circuiti T7-regolato, si può immaginare la produzione di biomolecole complesse in un ambiente user-controllabile tramite un mix di nativo E. promotori coli e meccanismi di trascrizione e regolazione esogena in dotazione. Senza limitazioni di divisione cellulare e il metabolismo, variabilità nei circuiti sintetici come il repressilator o in multistrato vie metaboliche come quelle che producono artemisinina può essere ridotta o meglio compresa. 30,31 Noi have utilizzato questi vantaggi per implementare interruttori genetici, nonché per capire sigma fattore sequestro 9,32 Tale tecnologia può anche costituire la spina dorsale del &quot;minimal&quot; o le celle &quot;artificiali&quot; -. piccola, ben caratterizzato e autosufficienti unità incarnata estratto. 33,34 In definitiva, ci aspettiamo usi immediati di questo sistema di libera espressione di cellule endogene come un ambiente di prototipazione per la biologia sintetica. Soprannominato il &quot;TX-TL tagliere biomolecolare&quot;, il sistema di espressione cell-free offre un ambiente controllabile in cui in ultima analisi, circuiti sintetici destinati in vivo l&#39;espressione può subire turni di prototipazione - cicli di test sul plasmide di base, lineare, o DNA sintetizzati chimicamente, seguita mediante analisi e la modifica rapida. Turni prototipazione possono essere aiutati da modelli matematici predittivi attualmente in fase di sviluppo. Rimuovendo la clonazione e manipolazione vivo per i circuiti non definitive, prevediamo ingegneritempi di ciclo gneria per essere ridotto a 1-3 giorni invece dello standard attuali settimane.

A correction to Figure 5's legend has been made for the article <a href="http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free">Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology</a>. Method 1 and method 2 have been switched.Z
The figure legend was update from:
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1 nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 1) or post-processed with a QiaQuick PCR purification kit (Purification method 2).
to
Figure 5. Effects of DNA solution on expression efficiency. a) Comparison of two different purification methods for processing plasmids. 1
nM of pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 is prepared using only a QiaPrep Spin Miniprep Kit (Purification method 2) or post-processed
with a QiaQuick PCR purification kit (Purification method 1).

A correction to Figure 5's legend has been made for the article Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Method 1 and method 2 have been switched.

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

La tecnologia libera espressione-Cell ha iniziato nel 1950 come puramente traslazionale, avanzando anni più tardi a comprendere i meccanismi di trascrizione-traduzione accoppiati con T7 DNA del batteriofago. 11,12 Da allora, sono stati compiuti numerosi sforzi per ottimizzare la creazione di estratto cellulare grezzo (o E . estratto coli S30). 13,14 Queste ottimizzazioni includono prolungare cell-free sintesi proteica attraverso la rigenerazione dell&#39;ATP o modifiche di deformazione, e riducendo i tempi ei costi di protocollo. 15-17 esistono sistemi di espressione cell-free alternativi che uso ricostituito componenti in sostituzione di greggio estratto di cellule per l&#39;espressione. 5 Sia estratto cellulare grezzo e metodi di ricostituzione sono state sviluppate per uso commerciale. Con l&#39;avvento della biologia sintetica, vi è una maggiore necessità di una piattaforma ben caratterizzato per provare ed esprimere i moduli biologici ingegnerizzati e circuiti. 18,19 Questa piattaforma deve essereversatile, ben caratterizzato, semplice da manipolare, e focalizzato sui componenti forniti dall&#39;utente. Nonostante sia sviluppato mezzo secolo prima, sistemi acellulari basano su E. coli intrinsecamente condividere questi requisiti, in quanto sono una rappresentazione semplificata in vitro dei processi cellulari senza la complessità di crescita e metabolismo. Inoltre, tutte le conoscenze fondamentali da lavoro in vivo su E. coli si applica facilmente a E. sistemi cell-free coli. Anche se i sistemi di espressione cell-free possono avere applicazioni in biologia sintetica, ad oggi l&#39;obiettivo della maggior parte dei sistemi di espressione cell-free è stata la massimizzazione delle proteine ​​e la resa metabolita. Questo viene ottenuto usando batteriofago T7 trascrizione di sequenze guidati dai promotori T7. 20 Anche se l&#39;espressione è efficiente e robusto, questi sistemi hanno uno scopo altamente specializzato. Metodi di regolazione delle cellule sono limitate, i modelli di DNA target deve essereriprogettato per includere i promotori T7, e alcune sequenze come i complessi ribosomali non possono essere trascritti e assemblati. 21,22 attuali sistemi di espressione cell-free non sono in grado di mantenere rendimenti elevati preservando meccanismi regolatori endogeni, la versatilità necessaria per la biologia sintetica. Abbiamo sviluppato un E. endogeno coli sistema di espressione cell-free che conserva l&#39;efficienza della espressione proteica dimostrata dai sistemi precedenti, ma aggiunge ulteriore versatilità, consentendo espressione e la regolazione basato su entrambi (T7 o altro) meccanismi endogeni ed esogeni. Il protocollo qui descritto si basa inizialmente su Kigawa et al. (2004) e Liu et al. (2005), ma ha modifiche significative. Utilizza Mg e K-glutammato over-Mg e K-acetato per una maggiore efficienza, rimuove 2-mercaptoetanolo, e lisa le cellule utilizzando un tallone-beater. 17,23,24 Bead-pestaggio è scelto sopra omogeneizzazione,metodi basati pressione, o sonicazione per il suo costo inferiore e rendimenti paragonabili ai sistemi concorrenti. 23 acido 3-fosfoglicerico (3-PGA) è usato come fonte di energia come è stato trovato che invia rese proteiche superiori rispetto alla creatina fosfato e fosfoenolpiruvato. 4,25 Il nostro sistema può produrre fino a 0,75 mg / ml di proteina reporter utilizzando un promotore a base sigma70-con gli operatori lambda-fago o un promotore T7-driven, simile a rese da altri sistemi commerciali. 4,6 Cinque giorni sono tenuti a produrre tutti i reagenti necessari (Figura 1). Inoltre, fornisce una riduzione dei costi del 98% rispetto ai sistemi comparabili commerciali cell-free - Costi per materie sono $ 0,11 per 10 microlitri di reazione, che sale a 0,26 dollari, con manodopera inclusa (Figura 2).

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>2xYT</td>
 <td>MP biomedicals</td>
 <td>3012-032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3-PGA</td>
 <td>Sigma-Aldrich</td>
 <td>P8877</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A8937</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bacto-agar</td>
 <td>BD Diagnostics</td>
 <td>214010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bead-beating tubes (polypropylene microvials)</td>
 <td>BioSpec</td>
 <td>522S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Beads, 0.1mm dia.</td>
 <td>BioSpec</td>
 <td>11079101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BL21 Rosetta 2 E. coli strain</td>
 <td>Novagen</td>
 <td>71402</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bradford BSA Protein Assay Kit</td>
 <td>Bio-rad</td>
 <td>500-0201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cAMP</td>
 <td>Sigma-Aldrich</td>
 <td>A9501</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C1919</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CoA</td>
 <td>Sigma-Aldrich</td>
 <td>C4282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CTP</td>
 <td>USB</td>
 <td>14121</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cuvettes, 1.5ml</td>
 <td>Fisher</td>
 <td>14-955-127</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Sigma-Aldrich</td>
 <td>D0632</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Folinic acid</td>
 <td>Sigma-Aldrich</td>
 <td>F7878</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GTP</td>
 <td>USB</td>
 <td>16800</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H6147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>K-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>G1149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mg-glutamate</td>
 <td>Sigma-Aldrich</td>
 <td>49605</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro Bio-Spin Chromatography Columns</td>
 <td>Bio-Rad</td>
 <td>732-6204</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NAD</td>
 <td>Sigma-Aldrich</td>
 <td>N6522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc 384-well optical bottom plates</td>
 <td>Thermo-Scientific</td>
 <td>142761</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nunc sealing tape</td>
 <td>Thermo-Scientific</td>
 <td>232701</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PEG-8000</td>
 <td>Promega</td>
 <td>V3011</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate dibasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8584</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium phosphate monobasic solution</td>
 <td>Sigma-Aldrich</td>
 <td>P8709</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RTS Amino Acid Sampler</td>
 <td>5 Prime</td>
 <td>2401530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit)</td>
 <td>Thermo-Scientific</td>
 <td>66382</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spermidine</td>
 <td>Sigma-Aldrich</td>
 <td>85558</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base</td>
 <td>Fischer</td>
 <td>BP1521</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA (from E. coli)</td>
 <td>Roche Applied Science</td>
 <td>MRE600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UTP</td>
 <td>USB</td>
 <td>23160</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1L Centrifuge Bottle</td>
 <td>Beckman-Coulter</td>
 <td>A98813</td>
 <td>This is specific for Avanti J-series; obtain equivalent size for centrifuge in use.</td>
 </tr>
 <tr>
 <td>4L Erlenmeyer Flask</td>
 <td>Kimble Chase</td>
 <td>26500-4000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Avanti J-26XP Centrifuge</td>
 <td>Beckman-Coulter</td>
 <td>393127</td>
 <td>Or 1L-capable centrifuge equivalent.</td>
 </tr>
 <tr>
 <td>Forma 480 Orbital Shaker</td>
 <td>Thermo Scientific</td>
 <td>480</td>
 <td>Or chest-size 6x4L shaker equivalent.</td>
 </tr>
 <tr>
 <td>JLA-8.1000 Rotor</td>
 <td>Beckman-Coulter</td>
 <td>363688</td>
 <td>Or 1L-capable, 5000 x g rotor equivalent for centrifuge.</td>
 </tr>
 <tr>
 <td>Mini-Beadbeater-1</td>
 <td>BioSpec</td>
 <td>3110BX</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
Supplemental Material 1. Recipes for Items. 
Chloramphenicol, 34 mg/ml: Prepare 0.51 g chloramphenicol and add ethanol to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
2xYT+P+Cm agar plate: Prepare 1.24 g 2xYT, 1.6 ml potassium phosphate dibasic solution @ 1 M, 0.88 ml potassium phosphate monobasic solution @ 1 M, 0.6 g agar, and water to 40 ml. Autoclave. Let cool to 50 &deg;C and add 40 &mu;l Cm. Aliquot 25 ml into a 100x15 mm petri dish, and let cool for an hour. 
2xYT+P media: Prepare 124 g 2xYT, 160 ml potassium phosphate dibasic solution @1 M, 88 ml potassium phosphate monobasic solution @ 1 M, and water to 4 L. Aliquot out into 2x1.88 L and 0.24 L. Autoclave. 
Tris base, 2 M: Prepare 60.57 g Tris base and water to 250 ml. Sterilize, store at RT for later use. 
DTT, 1 M: Prepare 2.31 g DTT and water to 15 ml. Filter sterilize (0.22 &mu;M), aliquot to 1 ml tubes, store at -20 &deg;C for later use. 
S30A buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, 50 ml Tris at 2M, acetic acid (to pH 7.7), and water to 2 L. Autoclave, store at 4 &deg;C, add 4 ml 1 M DTT before use. 
S30B buffer: Prepare 10.88 g Mg-glutamate and 24.39 g K-glutamate, Tris at 2 M (to pH 8.2), and water to 2 L. Autoclave, store at 4 &deg;C, add 2 ml 1 M DTT before use. 
HEPES: Prepare 1.91 g HEPES (MW 238.21), KOH (to pH 8), and water to 4 ml. 
tRNA: Prepare 30 mg of tRNA and water to 600 &mu;l. 
CoA: Prepare 30 mg of CoA (MW 767.53) and water to 600 &mu;l. 
NAD: Add 34.83 mg of NAD (MW 663.43), Tris at 2 M (to pH 7.5-8), and water to 300 &mu;l. (Add 27 &mu;l of Tris at 2 M to bring the solution to pH 7.5-8). 
cAMP: Add 42.80 mg of cAMP (MW 329.22), Tris at 2 M (to pH 8), and water to 200 &mu;l. (Add 73 &mu;l of Tris at 2 M to bring the solution to pH 8). 
Folinic Acid (33.9 mM): To 20 mg of solid folinic acid calcium salt (MW 511.5), add 1.15 ml water. 
Spermidine: Prepare 23.55 &mu;l of spermidine (MW 145.25) and water to 150 &mu;l. Prepare at room temperature after melting briefly at 37 &deg;C. 
3-PGA: Add 1.03 g of 3-PGA (MW 230.02), Tris at 2 M (to pH 7.5), and water to 3.2 ml. (Add 1.73 ml of Tris at 2 M to bring the solution to pH 7.5). 
Nucleotide Mix: Add 145 mg of ATP dipotassium salt dihydrate (MW 619.4), 133 mg of GTP disodium salt (MW 567.14), 79.4 mg of CTP disodium salt dihydrate (MW 563.16), 82.6 mg of UTP trisodium salt dihydrate (MW 586.12), KOH at 15% dilution (to pH 7.5), and water to 1.5 ml. (Add 353 &mu;l of KOH at 15% dilution to bring the solution to pH 7.5). 
Supplemental Material 2. Bradford Assay.
<ol>
<li>Remove Bradford agent from 4 &deg;C and set at room temperature.</li>
<li>Prepare 50 &mu;l BSA Standard at 1 mg/ml and at 0.1 mg/ml.</li>
<li>Prepare 40 &mu;l 20x dilution of extract from step 1.47.</li>
<li>Add 800 &mu;l water to 7 cuvettes. </li>
<li>Prepare standard cuvettes for 0 mg/ml, 1 mg/ml (10 &mu;l 0.1 mg/ml BSA), 2 mg/ml (20 &mu;l 0.1 mg/ml BSA), 4 mg/ml (4 &mu;l 1 mg/ml BSA), 6 mg/ml (6 &mu;l 1 mg/ml BSA).</li>
<li>Prepare experimental cuvettes for 2 &mu;l of sample and 4 &mu;l of sample.</li>
<li>Add 200 &mu;l of Bradford agent to each cuvette and mix well by pipetting. Incubate at room temperature for at least 10 min.</li>
<li>Produce standard curve at OD 595nm using cuvettes from step 6.5. Reject standard curve if r2 &lt; 0.95.</li>
<li> Determine extract concentration at OD 595nm using cuvettes froms tep 6.6.</li>
</ol>
Supplemental Material 3. Buffer calibration spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank">See TXTL_e(template)_calibration_JoVE.xlsx</a>.
 
Supplemental Material 4. Cell-free expression run spreadsheet. 
<a href="http://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">See TXTL _JoVE.xlsx</a>.
 </td>
 </tr>
 </tbody>
</table>

protocols for implementing an escherichia coli based tx-tl cell-free expression system for synthetic biology

Sistemi di espressione acellulari ideale può teoricamente emulare un ambiente cellulare in vivo in una piattaforma controllata in vitro. 1 Questo è utile per esprimere proteine ​​e circuiti genetici in modo controllato e per fornire un ambiente di prototipazione per biologia sintetica. 2,3 Per raggiungere il secondo obiettivo, i sistemi di espressione acellulari che conservano endogena Escherichia coli meccanismi di trascrizione-traduzione sono in grado di riflettere più accuratamente dinamiche cellulari in vivo rispetto a quelle a base di T7 RNA polimerasi trascrizione. Descriviamo la preparazione e l&#39;esecuzione di un efficiente E. endogeno basato coli trascrizione-traduzione (TX-TL) sistema di espressione senza cellula in grado di produrre quantità equivalenti di proteine ​​come sistemi basati T7 a una riduzione dei costi del 98% a sistemi commerciali simili. 4,5 La preparazione di tamponi e estratto cellulare grezzo sono descritto, così come l&#39;esecuzione di untre tubo di reazione TX-TL. L&#39;intero protocollo richiede cinque giorni di tempo per preparare e produce abbastanza materiale per un massimo di 3000 singole reazioni in una preparazione. Una volta preparato, ogni reazione prende meno di 8 ore dal setup per la raccolta e l&#39;analisi dei dati. Meccanismi di regolazione e trascrizione esogeno di E. coli, come repressori lac / Tet e T7 RNA polimerasi, potrà essere integrata. 6 immobili endogeni, come l&#39;mRNA e la velocità di degradazione del DNA, può anche essere adeguato. 7 Il sistema di libera espressione di cellule TX-TL è stata dimostrata per la grande montaggio scala circuito, esplorando fenomeni biologici, e l&#39;espressione di proteine ​​sotto le due promotori-T7 e endogeni. 6,8 modelli matematici di accompagnamento sono disponibili. 9,10 Il sistema risultante ha applicazioni uniche nella biologia sintetica come un ambiente di prototipazione, o &quot;TX- TL biomolecolare breadboard. &quot;

Abbiamo presentato un protocollo di cinque giorni per la preparazione di un Escherichia coli endogeno basato sistema di espressione acellulare TX-TL. Una cronologia esempio per la creazione dei reagenti - estratto di cellule greggio e tampone - può essere trovato in Figura 1. Una volta creati, questi possono essere conservati a -80 ° C per un massimo di un anno. Dopo i reagenti sono creati, setup sperimentale e l&#39;esecuzione può essere fatto in meno di 8 ore. Inoltre, abbiamo ottimizzato le condizioni di espressione del sistema di espressione acellulare TX-TL. Altre aggiunte forniti dall&#39;utente, come tamponi o soluzioni di DNA, devono essere calibrati per la tossicità anticipo. Ad esempio, diversi metodi di elaborazione dei risultati plasmidi di espressione differente dovuto contenuto di sale. Abbiamo anche testato l&#39;effetto di tampone di eluizione Tris-Cl sull&#39;efficienza reazione (Figura 5). Un esempio di calibrazione grezzo estratto cellulare, riferendosi passo 4,1-4,9, è mostratoin figura 4a. In generale, i nostri esperimenti mostrano che l&#39;estratto cellulare grezzo è più sensibile ai livelli di Mg-glutammato, seguiti da livelli di K-glutammato. Per dimostrare il sistema di libera espressione di cellule, abbiamo costruito e testato un ciclo di feedback negativo in base a tet repressione. 26 (Figura 6). Nel sistema di espressione cell-free, la stessa corsa circuito con e senza ATC mostra un end-point cambiamento di espressione di 7 volte di deGFP giornalista dopo otto ore di espressione. Anche se questo esperimento non richiede induttori globali o repressori, se necessario, possono essere aggiunti in &quot;Mix Preparazione Maestro&quot;. <img alt="Figura 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50762/50762fig1highres.jpg" src="/files/ftp_upload/50762/50762fig1.jpg" /> Figura 1. Timeline di estratto di cellule greggio, soluzione di aminoacidi, e la preparazione soluzione energetica. Timelin A cinque giorni<html> e per l&#39;esecuzione tipica del protocollo è dato sopra, ottimizzato per incubazioni overnight e fasi di lavoro durante il giorno. <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig2highres.jpg" src="/files/ftp_upload/50762/50762fig2.jpg" /> Figura 2. Costi e analisi di espressione di competere estratti cellulari greggio. a) Ripartizione dei costi di manodopera e dei materiali del sistema di libera espressione di cellule TX-TL. Sulla base dei costi dei reagenti come di Dicembre 2012, e il costo del lavoro di $ 14 all&#39;ora. B) Confronto dei costi di sistema libera espressione di cellule TX-TL vs altri sistemi commerciali. I costi sono ripartiti per ml, anche se i volumi di reazione possono variare per kit. C) Confronto tra TX-TL cell-free sistema di espressione del rendimento rispetto ad altri sistemi commerciali. Resa espressione proteica determinata dalle norme produttore./ 50762fig2large.jpg &quot;target =&quot; _blank &quot;&gt; Clicca qui per ingrandire la figura. <img alt="Figura 3" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig3highres.jpg" src="/files/ftp_upload/50762/50762fig3.jpg" /> Figura 3. Caricamento e lavorazione di un tubo tallone-pestaggio. a) Dimostrazione della corretta viscosità della soluzione di cellule-tallone. Soluzione Cell-tallone avrà una viscosità dipende da molti fattori, tra cui quantità di tampone S30A aggiunto, la quantità di perline aggiunti, e il tempo trascorso sul ghiaccio. B) Caricamento di tubo tallone battendo prima breve centrifugazione tavolo. La centrifugazione rimuove le bolle accumulate durante il caricamento. C) Bolle emersione dopo tavolo centrifugazione. La dimensione delle bolle varia, possono essere spuntato o rimosso utilizzando un puntale d) completamente riempito tubo tallone battere prima di tappatura.. Un menisco si forma nel tallone battendo tube, e il tappo ha abbastanza per coprire e causare piccole quantità per riempire troppo. e) apparecchio filtrante correttamente caricato. Questi possono essere riutilizzati. F) Confronto correttamente vs tubo tallone battere correttamente elaborato. Il tubo sulla sinistra è un tubo ben-beat - è dotato di un piccolo e ben delineato strato superiore, e molto chiaro surnatante. Il tubo a destra è ottimale, in base al formato grande, secondo strato nebuloso e il surnatante nebuloso. Tubi che sono subottimale non dovrebbero essere sottoposti a ulteriori elaborazioni. <img alt="Figura 4" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig4highres.jpg" src="/files/ftp_upload/50762/50762fig4.jpg" /> Figura 4. Proprietà di preparati estratto grezzo. a) curva di taratura tipici per estratto cellulare grezzo. Estratto grezzo è calibrato per ulteriori Mg-glutammato, K-glutammato, e livelli DTT, in questo ordine. Viene mostrato endpoint fluorescence dopo 8 ore, così come velocità massima di produzione di proteine ​​sulla base di una media mobile a 12 minuti. Sulla base di queste trame, un intervallo accettabile di ulteriori Mg-glutammato è di 4 mm, K-glutammato è 60-80 mm, DTT è 0-3 mm. Si noti che ogni estratto grezzo deve essere calibrato in modo indipendente per queste tre variabili. B) Variazione dalle preparazioni estratto. È mostrato Endpoint fluorescenza di due estratti grezzi preparati in date diverse, le barre di errore sono 1 deviazione standard da tre piste indipendenti in giorni diversi. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 5" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig5highres.jpg" src="/files/ftp_upload/50762/50762fig5.jpg" /> Figura 5. Effetti della soluzione di DNA sull&#39;efficienza espressione. a) Confronto tra due diversi depurazionemodalità di trattamento dei plasmidi. 1 nM di pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 viene preparata utilizzando solo un kit QIAprep Spin Miniprep (metodo di purificazione 1) o post-elaborata con un kit di purificazione QIAquick PCR (metodo di purificazione 2). Puo &#39;endpoint fluorescenza dopo 8 ore, così come velocità massima di produzione di proteine ​​sulla base di una media mobile a 12 minuti. Le barre di errore sono 1 deviazione standard da quattro piste indipendenti in giorni diversi. B) Effetto del tampone di eluizione (Tris-Cl). Diverse concentrazioni di Tris-Cl sono confrontati in una reazione espressione cell-free base all&#39;espressione di 1 nM di pBEST-OR2-OR1-Pr-UTR1-deGFP-T500. Le concentrazioni indicate sono concentrazioni finali di Tris-Cl nella reazione; tampone di eluizione utilizzato è 10 mM Tris-Cl. Le barre di errore sono 1 deviazione standard da tre piste indipendenti in giorni diversi. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig5large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <p class="jove_content" fo:keep-together.within-page = &quot;always&quot;&gt; <img alt="Figura 5" fo:content-width="6in" fo:src="/files/ftp_upload/50762/50762fig6highres.jpg" src="/files/ftp_upload/50762/50762fig6.jpg" /> Figura 6. Esempio di run TX-TL di un ciclo di feedback negativo. a) la configurazione di esempio di una reazione esecuzione cell-free. Prove &quot;a&quot; &quot;off&quot; stato del ciclo di feedback negativo rispetto, con controlli positivi e negativi. B) plasmide mappa di feedback negativo. C) I risultati rappresentativi. Dati riflette esperimento in a) e b), con controllo negativo sottratto dal segnale. Circuito genetico mostrato in insert. Le barre di errore sono 1 deviazione standard da tre piste indipendenti in giorni diversi. <a href="https://www.jove.com/files/ftp_upload/50762/50762fig6large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <table border="1"><tbody><tr><td> Nome </td><td> Concentrazione </td><td> Importo </td><td> Sterilizzazione </td><td> Note </td></tr><tr><td> Cloramfenicolo (Cm) </td><td> 34 mg / ml in etanolo </td><td> 1 ml </td><td> Filtro sterilizzare (0,22 micron) </td><td> Può essere fatto in grandi volumi conservati a -20 ° C per un uso successivo. </td></tr><tr><td> 2xYT + P + piastra Cm agar </td><td> 31 g / L 2xYT, 40 mM potassio fosfato bibasico, 22 mM fosfato di potassio monobasico, 34 mg / ml di cloramfenicolo </td><td> 1 piastra </td><td> Autoclave </td><td></td></tr><tr><td> 2xYT + P supporti </td><td> 31 g / L 2xYT, 40 mM potassio fosfato bibasico, 22 mM fosfato di potassio monobasico </td><td> 4 L </td><td> Autoclave </td><td></td></tr></tbody></table> Tabella 1. Reagenti per giorno 1 del protocollo estratto cellulare grezzo. <table border="1"><tbody><tr><td> Nome </td><td> Concentrazione </strong&gt; </td><td> Importo </td><td> Sterilizzazione </td><td> Note </td></tr><tr><td> Tris base </td><td> 2 M </td><td> 250 ml </td><td> Sterilizzare Filter (0,22 micron) o in autoclave </td><td> Può essere conservato a temperatura ambiente. </td></tr><tr><td> DTT </td><td> 1 M </td><td> 6 ml </td><td> Filtro sterilizzare (0,22 micron) </td><td> Può essere fatto in grandi volumi e conservati a -20 ° C per un uso successivo. </td></tr><tr><td> Tampone S30A </td><td> 14 mM Mg-glutammato, 60 mM K-glutammato, 50 mM Tris, pH 7.7 </td><td> 2 L </td><td> Autoclave </td><td> Per raggiungere pH 7.7, titolare con acido acetico. Aggiungi DTT a 2 mm di concentramento finale appena prima dell&#39;uso. Conservare a 4 ° C. </td></tr><tr><td> Tampone S30B </td><td> 14 mM Mg-glutammato, 60 mm K-glutammato, ~ Tris 5 mM, pH 8,2 </td><td> 2 L </td><td> Autoclave </td><td> Per raggiungere pH 8,2, titolare con 2MTris. Aggiungi DTT a 1 mM concentrazione finale appena prima dell&#39;uso. Conservare a 4 ° C. </td></tr></tbody></table> Tabella 2. Reagenti per il giorno 2 del protocollo estratto cellulare grezzo. <table border="1"><tbody><tr><td></td><td colspan="4"> Falco </td></tr><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td></tr><tr><td> Vuoto 50 ml Falcon (g) </td><td></td><td></td><td></td><td></td></tr><tr><td> 50 ml Falcon con pellet (g) </td><td></td><td></td><td></td><td></td></tr><tr><td> Massa Pellet (50 ml Falcon con pellet - vuoto 50 ml Falcon) (g) </td><td></td><td></td><td></td><td></td></tr><tr><td> S30A volume di tampone di aggiungere (massa pellet * 0,9) (ml) </td><td></td&gt; <td></td><td></td><td></td></tr><tr><td> Massa totale di perline per aggiungere (massa pellet * 5.0) (g) </td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabella 3. Tampone S30A e calcolatrice massa tallone, per il giorno 3 del protocollo estratto cellulare grezzo. <table border="1"><tbody><tr><td> Nome </td><td> Concentrazione </td><td> Importo </td><td> Sterilizzazione </td><td> Note </td></tr><tr><td> HEPES </td><td> 2 M, pH 8 </td><td> 4 ml </td><td> Nessuno </td><td> Per raggiungere pH 8, titolare con KOH. </td></tr><tr><td> Nucleotide Mix </td><td> , 94 mM CTP 156 mM ATP e GTP e UTP, pH 7.5 </td><td> 1,5 ml </td><td> Nessuno </td><td> Per raggiungere pH 7.5, titolare con KOH. </td></tr><tr><td> tRNA </td><td> 50 mg / ml </td><td> 600 microlitri </td><td> Nessuno </td><td></td></tr><tr><td> CoA </td><td> 65 mm </td><td> 600 microlitri </td><td> Nessuno </td><td></td></tr><tr><td> NAD </td><td> 175 mM, pH 7,5-8 </td><td> 300 ml </td><td> Nessuno </td><td> Per raggiungere pH 7.5-8, titolare con Tris a 2 M. </td></tr><tr><td> cAMP </td><td> 650 mM, pH 8 </td><td> 200 ml </td><td> Nessuno </td><td> Per raggiungere pH 8, titolare con Tris a 2 M. </td></tr><tr><td> Acido folinico </td><td> 33.9 mM </td><td> 300 ml </td><td> Nessuno </td><td> Anche se sono necessari solo 300 ml, ricetta supplementare è di 1.15 ml. </td></tr><tr><td> Spermidina </td><td> 1 M </td><td> 150 microlitri </td><td> Nessuno </td><td> Conservare a 4 ° C, calore a 37 ° C per fondere. </td></tr><tr><td> 3-PGA </td><td> 1.4 M, pH 7.5 </td><td> 3.2 ml </td><td> Nessuno </td><td> Per raggiungere pH 7.5, titolare con Tris a 2 M. </td></tr></tbody></table> Tabella 4. Reagenti per prepararsi per il protocollo Energy Solution. Supplemental Materiale 1. Ricette per Articoli. Cloramfenicolo, 34 mg / ml: Preparare 0,51 g cloramfenicolo e aggiungere etanolo a 15 ml. Sterilizzare Filter (0,22 micron), aliquota da 1 ml tubi, conservare a -20 ° C per un uso successivo. 2xYT + P + piastra Cm agar: Preparare 1,24 g 2xYT, 1,6 ml di soluzione di potassio fosfato bibasico @ 1 M, 0,88 ml di potassio fosfato monobasico @ 1 M, 0,6 g di agar, e l&#39;acqua a 40 ml. Autoclave. Lasciate raffreddare a 50 ° C e aggiungere 40 microlitri Cm. Aliquota 25 ml in una piastra di Petri di 100 x 15 mm, e lasciate raffreddare per un&#39;ora. 2xYT + P supporti: Preparare 124 g 2xYT, 160 ml di fosfato di potassio dibasico @ 1 M, soluzione di 88 ml di potassio fosfato monobasico @ 1 M, e acqua per 4 L. Aliquot fuori in 2 x 1,88 L e 0,24 L. Autoclave. Tris base, 2 M: Preparare 60,57 g di base Tris e acqua a 250 ml. Sterilizzare, conservare a temperatura ambiente per un uso successivo. DTT, 1 M: Preparare 2.31 ​​g DTT e acqua a 15 ml. Sterilizzare Filter (0,22 micron), aliquota da 1 ml tubi, conservare a -20 ° C per un uso successivo. Tampone S30A: Preparare 10.88 g Mg-glutammato e 24,39 g K-glutammato, 50 ml Tris a 2M, acido acetico (a pH 7,7), e acqua per 2 L. Autoclave, conservare a 4 ° C, aggiungere 4 ml di 1 M DTT prima dell&#39;uso. Tampone S30B: Preparare 10.88 g Mg-glutammato e 24,39 g K-glutammato, Tris a 2 M (pH 8.2), e acqua per 2 L. Autoclave, conservare a 4 ° C, aggiungere 2 ml di 1 M DTT prima dell&#39;uso. HEPES: Preparare 1.91 g HEPES (MW 238,21), KOH (a pH 8), e acqua a 4 ml. tRNA: Preparare 30 mg di tRNA e acqua a 600 ml. CoA: Preparare 30 mg di CoA (MW 767,53) e l&#39;acqua a 600 ml. NAD: Aggiungere 34.83 mg di NAD (MW 663,43), Tris a 2 M (a pH 7,5-8), e acqua a 300 ml. (Aggiungere 27 ml di Tris a 2 M per portare la soluzione a pH 7,5-8). cAMP: Aggiungere 42.80 mg di cAMP (MW 329,22), Tris a 2 M (a pH 8), e acqua di 200 microlitri. (Aggiungere 73 ml di Tris a 2 M per portare la soluzione a pH 8). Acido folinico (33,9 mm): A 20 mg di solido sale di calcio di acido folico (511,5 MW), aggiungere 1,15 ml di acqua. Spermidina: Preparare 23.55 ml di spermidina (MW 145,25) e acqua a 150 ml. Preparare a temperatura ambiente dopo la fusione brevemente a 37 ° C. 3-PGA: Aggiungere 1,03 g di 3-PGA (MW 230,02), Tris a 2 M (pH 7,5), e acqua a 3,2 ml. (Aggiungere 1,73 ml di Tris a 2 M per portare la soluzione a pH 7,5). Nucleotide Mix: Aggiungere 145 mg di ATP dipotassio diidrato sale (619,4 MW), 133 mg di GTP sale disodico (MW 567,14), 79,4 mg di CTP diidrato sale disodico (MW 563,16), 82,6 mg di diidrato UTP sale trisodico (MW 586,12) , KOH al 15% di diluizione (a pH 7,5), e acqua a 1,5 ml. (Aggiungere 353 ml di KOH al 15% diluizione per portare la soluzione a pH 7,5). Supplemental materiale 2. Bradford Assay. <ol><li> Rimuovere agente Bradford da 4 ° C e impostare a temperatura ambiente. </li><li> Preparare 50 ml BSA standard a 1 mg / ml ed a 0.1 mg / ml. </li><li> Preparare 40 ml 20x diluizione dell&#39;estratto dal punto 1.47. </li><li> Aggiungere 800 microlitri di acqua a 7 cuvette. </li><li> Preparare cuvette standard per la 0 mg / ml, 1 mg / ml (10 ml 0,1 mg / ml BSA), 2 mg / ml (20 ml 0,1 mg / ml BSA), 4 mg / ml (4 microlitri 1 mg / ml BSA) , 6 mg / ml (6 ml 1 mg / ml BSA). </li><li> Preparare cuvette sperimentali per 2 ml di campione e 4 ml di campione. </li><li> Aggiungere 200 ml di agente Bradford ad ogni provetta e mescolare bene by pipettaggio. Incubare a temperatura ambiente per almeno 10 min. </li><li> Produrre curva standard a OD 595 nm utilizzando cuvette dal punto 6.5. Rifiuta curva standard se r 2 &lt;0,95. </li><li> Determinare la concentrazione di estratto di OD a 595 nm utilizzando cuvette dal punto 6.6. </li></ol> Materiale supplementare 3. Buffer foglio di calibrazione. <a href="https://www.jove.com/files/ftp_upload/50762/TXTL_e(template)_calibration_JoVE.xlsx" target="_blank">Vedere TXTL_e (template) _calibration_JoVE.xlsx</a> . Materiale supplementare 4. Libera espressione-Cell eseguito foglio di calcolo. <a href="https://www.jove.com/files/ftp_upload/50762/TXTL_JoVE.xlsx" target="_blank">Vedere TXTL _JoVE.xlsx</a> .

Watch this Scientific Journal Video about Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology at JoVE.com

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

Questo protocollo di cinque giorni delinea tutte le fasi, attrezzature e software supplementare necessario per la creazione e l&#39;esecuzione di un sistema di espressione TX-TL cell-free da zero efficiente Escherichia coli endogeno basato. Con i reagenti, il protocollo dura 8 ore o meno per impostare una reazione, raccogliere e elaborare i dati.

Protocolli per l&#39;implementazione di un<em&gt; Escherichia coli</em&gt; Sulla base TX-TL cellulare-Free Expression System per la biologia sintetica

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

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Biology

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

62.7K Views.  California Institute of Technology.  Questo protocollo di cinque giorni delinea tutte le fasi, attrezzature e software supplementare necessario per la creazione e l'esecuzione di un sistema di espressione TX-TL cell-free da zero efficiente Escherichia coli endogeno basato. Con i reagenti, il protocollo dura 8 ore o meno per impostare una reazione, raccogliere e elaborare i dati. 

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

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.

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina 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 ...

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Biologia

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

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

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

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocolli di selezione mutagenesi e funzionale per l&#39;evoluzione Regia di proteine ​​in 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 ...

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.

Throughput Screening elevati di attività Endoglucanase fungine 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.

Mappatura batteriche reti funzionali e sulle vie di<em&gt; Escherichia Coli</em&gt; Utilizzando matrici sintetiche genetiche

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 ...

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 Protocollo per la preparazione di elettrocompetenti<em&gt; Escherichia coli</em&gt; E<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

Rilevamento dal vivo<em&gt; Escherichia coli</em&gt; O157: H7 Celle di 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. ...

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 Espressione Proiezione di proteine ​​di membrana 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 ...

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.

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.

I benefici multiforme Protein Co-espressione 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 ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Sistemi HeLa cellule base libera espressione a manifestare<em&gt; Plasmodium</em&gt; Rhoptry Proteine

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

L&#39;incorporazione specifici residuo di non canonici amminoacidi in proteine ​​modello utilizzando un<em&gt; Escherichia coli</em&gt; Trascrizione-traduzione sistema non cellulare