אנו מודים Jongmin קים, דן סיגל-Gaskins, Anu Thubagere, וחנוך Yeung לסיוע התייעלות הפרוטוקול, וקלייר חן וברקלי לי לסיוע בשלבים המוקדמים של הפרויקט בשלבים. חומר זה מבוסס על נתמך בחלקו על ידי ההגנה מחקר מתקדם פרויקטים הסוכנות (DARPA / MTO) תכנית החיים יציקה, מספר חוזה HR0011-12-C-0065 (DARPA / CMO.ZZS נתמך גם על ידי מדען רפואי UCLA / קלטק העבודה מלגת תכנית אימונים ועל ידי משרד ההגנה, חיל אוויר Office של מחקר מדעי, ביטחון הלאומי, מדע והנדסה לתואר שני (NDSEG) מלגה, 32 168a CFR. הדעות והמסקנות הכלולות במסמך זה הן על דעת המחברים ולא צריך להתפרש כמייצג באופן רשמי מדיניות, במפורש או במשתמע, של הסוכנות לפרויקטי מחקר מתקדם הביטחון או ממשלת ארה&quot;ב.

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

מערכת ביטוי תא ללא TX-TL מבוסס חיידקי Escherichia אנדוגני שתוארה כאן היא תגובה שלושה צינור קלה לטווח שיכול לקחת פחות משמונה שעות מלהגדיר לאיסוף נתונים. התהליך של יצירת כל ריאגנטים דורש חמישה בסך הכל &#39;ימי זמן (עם דרישות עבודה משמעותיות על יום אחד בלבד), אבל מייצר תמצית גולמי ל3,000 תגובות וריאגנטים ל10,000 תגובות (איור 1), מה שהופך את החיץ. יתר על כן, תמצית גולמי וחומרים כימיים יציבים במשך שנה לפחות 1 ב -80 מעלות צלזיוס, דבר המאפשרים לשימושים מרובים של הכנה אחד קבלת חיץ. 4 ב0.11 דולרים ל10 תגובת μl (0.26 $ כוללים עבודה), עלויות הן 98% בהשוואה להשוואה מערכות מסחריות (איור 2). ישנן מספר מגבלות בלתי פתורות, עם זאת, למערכת. יעילות סוף כל הכנת תמצית תא גולמי יכולה להשתנות על סמך בקיאות משתמש ועל תנאים סביבתיים, אם כי לאוריאציה תשואת ypical היא בין 5-10% (איור 4). כתוצאה מכך, השתנות אצווה כדי אצווה בשני ביטוי נקודת הסיום ובדינמיקת ביטוי הן צפויות. וריאציות אלו צפויות להישאר עד התמצית מאופיינת או עד יצירת תמצית היא אוטומטית לחלוטין באופן מלא. אם מערכת הביטוי ללא תא משמשת לבצע ניסויים כמותיים רגישים, רצוי להפעיל את כל הניסויים עם אותה הקבוצה של תמצית תא גולמי. התשואה מתצווה אחת תמצית תא גולמי, על 3000 תגובות, צריכה להיות מספיק עבור קורסים ניסיוניים טיפוסיים. למרות שאנו חושדים וריאציה ניתן להסיר על ידי דרוג את ומיכון ההליך, ניסיונות כאלה היינו כרוכים בהשקעת משאבים משמעותית. בנוסף, למרות שרמות ביטוי נקודת סיום הן קלים באופן סביר על מנת לקבוע, עוד עבודה שצריכה לעשות בדינמיקת הבנה פנימית למערכת ללא תא. זה ידוע כי שניהם competitio המשאבהגבלת n ומשאבים יכולה להשפיע על דינמיקת ביטוי. לדוגמא, סיגמא אנדוגני המוגבל 70 יכול לגרום למשטר להרוות עם תבנית ה-DNA מוגבר לייצר פרופיל ביטוי מקביל לזה של דלדול נוקלאוטיד או חומצת אמינו. 9,27 עם זאת, דינמיקה לא צריך להיות מובנת במלוא לנצל את המערכת. לעלייה טהורה של תשואה, אופטימיזציה יכולה להיעשות על ידי גישות למידה מכונה. 28 שאלות של תחרות משאבים והגבלה ניתן לטפל על ידי מודלים מתמטיים לאמת באמצעות נתונים ניסיוניים. הפרוטוקול המובא כאן הוא מותאם לזן BL21-Rosetta2, אבל הוא להכליל את א &#39;אחרת זני חיידק. שינויים בBL21-Rosetta2, כגון הסרת פרוטאז גן הקידוד לון והתוספת של הגנים המקודדים tRNAs הנדיר, לאפשר לייצור חלבון מקסימאלי. יש לנו ניסיון בפרוטוקול עם שני זני תמצית אחרים - BL21 בלבד ונוקאאוט-BL21 trxAד מצא תשואת חלבון 50% פחות. אנו משערים כי תשואות דומות להקטין בעת ​​שימוש בזנים אחרים. שינויים אחרים בפרמטרים, כגון החלפת מדיום גידול 2xYT עבור LB ומרקים עשירים אחרים, הביאו לתשואת חלבון ירד. יש מערכות ביטוי תא ללא ניצול שני מכונות שעתוק-תרגום אנדוגני ואקסוגני ומנגנוני רגולציה יישומים רחבים בחלבון וגם ביטוי המטבוליט ובביולוגיה סינתטית. 3,29 במקום להיות מוגבל למעגלים מוסדרים T7, אפשר לדמיין הפקת ביומולקולות המורכבת בהגדרת משתמש לשליטה באמצעות שילוב של א &#39;ילידים יזמים coli ומנגנוני שעתוק ורגולציה מסופקים אקסוגני. ללא מגבלות של חלוקת תא וחילוף חומרים, השתנות במעגלים סינטטיים כגון repressilator או במסלולים מטבוליים מהונדס כגון ארטמיסינין הייצור אלה יכולים להיות מופחתים או להבין טובים יותר. 30,31 אנו havדואר משמש יתרונות אלה ליישם מתגים גנטיים, כמו גם להבין את תפיסת גורם סיגמא טכנולוגיה זו 9,32 יכולה גם מהווה את עמוד השדרה של תאים &quot;מלאכותיים&quot; &quot;מינימאלי&quot; או -. קטן, מאופיין היטב ויחידות גלומות עצמאית של תמצית. 33,34 סופו של דבר, אנו צופים שימושים מיידיים של מערכת ביטוי זה אנדוגני תא ללא כסביבת דיגום לביולוגיה סינתטית. כינוי &quot;קרש חיתוך biomolecular TX-TL,&quot; מערכת ביטוי התא ללא מספקת סביבה לשליטה בו מעגלים סינטטיים נועדו סופו של דבר לin vivo ביטוי יכול לעבור סיבובים של בניית אב טיפוס - מחזורים של בדיקות על פלסמיד הבסיסי, ליניארי, או דנ&quot;א synthetized כימי, ואחריו על ידי ניתוח ושינוי מהיר. יכולים להיות בעזרת סיבובים prototyping על ידי מודלים מתמטיים חזוי כרגע בשלבי פיתוח. על ידי הסרת שיבוט ובמניפולצית vivo למעגלים שאינם סופיים, אנו צופים engiזמני מחזור חלוצית ליופחתו ל 1-3 ימים במקום סטנדרטית &#39;השבועות הנוכחיים.

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

טכנולוגית ביטוי תא ללא החלה ב1950s כגרידא translational, קידום שנים מאוחר יותר על מנת להקיף מנגנוני שעתוק-תרגום בשילוב באמצעות DNA bacteriophage T7. 11,12 מאז, מאמצים רבים נעשו כדי לייעל את הקמתה של תמצית תא גולמי (או E . תמצית coli S30). 13,14 אופטימיזציות אלו כוללות הארכת סינתזת חלבון ללא תא דרך התחדשות ATP או שינויי מתח, וצמצום זמן פרוטוקול ועלות. 15-17 מערכות ביטוי תא ללא אלטרנטיביים קיימות שרכיבי שימוש מחדש במקום גולמי תא תמצית לביטוי. 5 שתי תמצית תא גולמי ושיטות הכינון מחדש פותחו לשימוש מסחרי. עם כניסתו של ביולוגיה סינתטית, יש צורך מוגבר פלטפורמה מאופיינת היטב כדי לבחון ולהביע את המודולים ביולוגיים מהונדסים ומעגלים. 18,19 פלטפורמה זו חייבת להיותתכליתי, מאופיין היטב, פשוט לתפעול, ומתמקד ברכיבים המסופק על ידי משתמש. למרות מפותח חצי מאה קודם לכן, מערכות תא ללא מבוססות על E. coli חולק באופן מהותי בדרישות אלה, כפי שהם פשוט יותר בייצוג מבחנה של תהליכים תאיים ללא המורכבות של צמיחה וחילוף חומרים. בנוסף, את כל הידע הבסיסי בעבודה מvivo על E. coli מתייחס בקלות לE. מערכות תא ללא coli. למרות שמערכות ביטוי תא ללא יכולות להיות יישומים בביולוגיה סינתטית, עד היום המטרה של רוב מערכות ביטוי תא ללא כבר מיצוי החלבון ותשואת המטבוליט. המטרה זו מושגת על ידי שימוש בשעתוק bacteriophage T7 של רצפים מונעים על ידי היזמים T7. 20 למרות שהביטוי הוא יעיל וחזק, מערכות אלו לשרת מטרה מאוד מיוחדת. שיטות רגולציה סלולרי הן מוגבלות, תבניות ה-DNA יעד חייבת להיותreengineered לכלול יזמים T7, ולא ניתן עיבד רצפים מסוימים, כגון מתחמי ריבוזומלי ומורכבים. 21,22 מערכות ביטוי תא ללא קיימות לא מצליחות לשמור על תשואות גבוהות תוך שמירה על מנגנוני ויסות אנדוגני, צדדיות הכרחי לביולוגיה סינתטית. פיתחנו ה אנדוגני מערכת תא ללא coli ביטוי שמשמרת את היעילות של ביטוי חלבון הודגמה על ידי מערכות קודמות, אך מוסיפה צדדיות נוסף על ידי המאפשר ביטוי ורגולציה מבוסס על שני במשק ומחוץ מנגנונים (T7 או אחרים). הפרוטוקול המתואר כאן מבוסס במקור על Kigawa et al. (2004) ויו et al. (2005), אבל יש שינויים משמעותיים. הוא מנצל Mg-ו-K-גלוטמט על Mg-ו-K-אצטט להתייעלות, מסיר 2-mercaptoethanol, וlyses תאים באמצעות חרוז-מקצף. 17,23,24 חרוזים מכות הוא נבחר על פני הומוגניזציה,שיטות המבוסס על לחץ, או sonication בשל עלותו נמוכה יותר ותשואות דומות למערכות מתחרות. 23 חומצת 3-phosphoglyceric (3-PGA) משמשת כמקור האנרגיה כפי שהוא נמצא לתת תשואות חלבון מעולים בהשוואה לקריאטין פוספט ו phosphoenolpyruvate. 4,25 המערכת שלנו יכולה לייצר עד 0.75 מ&quot;ג / מיליליטר חלבון כתב של שימוש גם אמרגן מבוסס sigma70 עם מפעילי מבדה-הפאג או אמרגן מונע T7, בדומה לתשואות ממערכות מסחריות אחרות. 4,6 חמישה ימים נדרש כדי לייצר את כל חומרים כימיים הנחוצים (איור 1). יתר על כן, הוא מספק הפחתת עלויות 98% בהשוואה למערכות דומות מסחריות תא ללא - עלויות חומר הם 0.11 דולרים ל10 תגובת μl, המתנשא ל -0.26 $ עם עבודה כלולה (איור 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>
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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>.
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protocols for implementing an escherichia coli based tx-tl cell-free expression system for synthetic biology

מערכות ביטוי תא ללא אידיאלית באופן תיאורטי יכולות לחקות סביבה תאית in vivo שבשליטה בפלטפורמת מבחנה. 1 זו שימושית לביטוי חלבונים ומעגלים גנטיים באופן מבוקר, כמו גם למתן סביבת דיגום לביולוגיה סינתטית. 2,3 כדי להשיג את המטרה האחרונה, מערכות ביטוי תא ללא שלשמר מנגנוני שעתוק-תרגום חיידקי Escherichia אנדוגני הם מסוגלים לשקף בצורה מדויקת יותר in vivo דינמיקה הסלולר יותר מאלו המבוססים על שעתוק RNA פולימראז T7. אנו מתארים את ההכנה וביצוע של א אנדוגני יעיל מערכת ביטוי תמלול בתרגום מבוסס coli (TX-TL) ללא תא שיכול לייצר כמויות שווה של חלבון כמו מערכות מבוססות-T7 ב98% הפחתת עלויות למערכות מסחריות דומות. 4,5 הכנת מאגרים ותמצית תא הגולמי הן תואר, כמו גם ביצועתגובת TX-TL שלושה צינור. כל הפרוטוקול לוקח חמישה ימים להכין ומניב מספיק חומר לעד 3000 תגובות בודדות בהכנה אחד. מוכן ברגע, כל תגובה לוקחת פחות מ -8 שעות מהתקנה לאיסוף וניתוח הנתונים. מנגנונים של רגולציה ואקסוגניים שעתוק לE. coli, כגון repressors אק / ט ופולימראז T7-RNA, ניתן להשלים. 6 נכסים אנדוגני, כגון ה-mRNA ושיעורי השפלה DNA, יכול גם להיות מותאם. 7 מערכת ביטוי התא ללא TX-TL הוכח עבור גדול הרכבה בקנה מידה במעגל, לחקור תופעות ביולוגיות, וביטוי של חלבונים תחת שני היזמים T7-ואנדוגני. 6,8 מודלים מתמטיים נלווים זמינים. 9,10 המערכת וכתוצאה מכך יש לו יישומים ייחודיים בביולוגיה סינתטית כסביבת prototyping, או &quot;TX- קרש חיתוך biomolecular TL. &quot;

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

פרוטוקול של חמישה ימים זה מתאר את כל השלבים, ציוד ותוכנה נוספת דרושה ליצירה והפעלת מערכת ביטוי תא ללא TX-TL מאפס חיידקי Escherichia אנדוגני יעילים מבוססים. עם חומרים כימיים, הפרוטוקול לוקח 8 שעות או פחות להגדרת תגובה, לאסוף, ולעבד נתונים.

פרוטוקולים ליישום<em&gt; חיידקי Escherichia</emמערכת&gt; בהתבסס TX-TL סלולרי ללא ביטוי של ביולוגיה סינתטית

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

62.8K Views.  California Institute of Technology.  פרוטוקול של חמישה ימים זה מתאר את כל השלבים, ציוד ותוכנה נוספת דרושה ליצירה והפעלת מערכת ביטוי תא ללא TX-TL מאפס חיידקי Escherichia אנדוגני יעילים מבוססים. עם חומרים כימיים, הפרוטוקול לוקח 8 שעות או פחות להגדרת תגובה, לאסוף, ולעבד נתונים. 

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.

Assay למדידת פעילות Escherichia coli ועין מתנהלת ליזין 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 ...

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.

בחירת הפרוטוקולים mutagenesis ו פונקציונאלי אבולוציה מכוונת של חלבונים א 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.

הקרנת תפוקה גבוהה של פעילות Endoglucanase פטרייתיים ב 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.

מיפוי רשתות פונקציונליות חיידקיים ומסלולים ב<em&gt; חיידקי Escherichia</em&gt; באמצעות מערכים גנטיים סינטטיים

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.

ראפיד פרוטוקול להכנת Electrocompetent<em&gt; חיידקי Escherichia</em&gt; ו<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

איתור של שידור חי<em&gt; חיידקי Escherichia</em&gt; O157: H7 תאים על ידי 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.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</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.

תועלת רבה פנים של Co-ביטוי חלבון ב<em&gt; Coli Escherichia</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.

מערכות ביטוי חופשי הנייד מבוסס הלה לביטוי של<em&gt; Plasmodium</em&gt; Rhoptry חלבונים

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.