نشكر Jongmin كيم، دان سيغال-Gaskins، أنو Thubagere، واينوك يونغ للمساعدة تبسيط البروتوكول، وكلير تشن لي وباركلي للمساعدة في المراحل الأولى من المشروع. ويستند هذه المواد على العمل بدعم جزئي من وكالة مشاريع أبحاث الدفاع المتقدمة (داربا / MTO) برنامج المعيشة مسابك، عدد العقد HR0011-12-C-0065 (معتمد داربا / CMO.ZZS أيضا من قبل العلماء في جامعة كاليفورنيا الطبي / معهد كاليفورنيا للتكنولوجيا برنامج الزمالة التدريبية من قبل وزارة الدفاع، وجهات النظر والاستنتاجات الواردة في هذه الوثيقة هي آراء المؤلفين، وينبغي ألا يفسر القوات الجوية مكتب البحث العلمي والعلوم الدفاع الوطني وخريج كلية الهندسة (NDSEG) زمالة، 32 CFR 168A. كما تمثل رسميا السياسات، إما صراحة أو ضمنا، من وكالة مشاريع البحوث المتقدمة الدفاع أو الحكومة الأمريكية.

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

والإشريكية القولونية الذاتية استنادا TX-TL نظام التعبير خالية من الخلايا الموصوفة هنا هو وسيلة سهلة للتشغيل أنبوب ثلاثة ردود الفعل التي يمكن أن تستغرق أقل من ثماني ساعات من وضع لجمع البيانات. عملية إنشاء جميع الكواشف يتطلب خمسة أيام الوقت الكلي (مع متطلبات العمل كبيرة في يوم واحد فقط)، ولكن تنتج استخراج النفط الخام ل3،000 ردود الفعل وردود الفعل الكواشف ل10،000 (الشكل 1) صنع العازلة. وعلاوة على ذلك، واستخراج النفط الخام والكواشف مستقرة لا يقل عن 1 سنة في -80 درجة مئوية، مما يسمح لاستخدامات متعددة من التحضير واحد صنع العازلة 4. في 0،11 $ لكل 10 رد فعل ميكرولتر (0،26 $ بما في ذلك العمل)، وتكاليف أقل 98٪ من مقارنة الأنظمة التجارية (الشكل 2). هناك بعض القيود التي لم تحل، ولكن، إلى النظام. كفاءة نهاية كل خلية إعداد مستخلص الخام يمكن أن تختلف على أساس الكفاءة المستخدم وعلى الظروف البيئية، على الرغم من راختلاف العائد ypical ما بين 5-10٪ (الشكل 4B). ونتيجة لذلك، وتقلب دفعة إلى دفعة في كل من التعبير نقطة النهاية وديناميكية في التعبير وينتظر أن تتم. ومن المرجح أن تظل هذه الاختلافات حتى يتميز استخراج بالكامل أو حتى يتم إنشاء آلية استخراج بالكامل. إذا تم استخدام نظام التعبير خالية من الخلايا لإجراء التجارب الكمي الحساسة، فإنه من المستحسن لتشغيل جميع التجارب مع نفس دفعة من استخراج الخلايا الخام. العائد من نفس واحدة استخراج الخام دفعة الخلية، حوالي 3000 ردود الفعل، ينبغي أن يكون كافيا لدورات تجريبية نموذجية. على الرغم من أن نشك يمكن إزالة الاختلاف من خلال زيادة وأتمتة الإجراءات، فإن مثل هذه المحاولات تنطوي على استثمار الموارد كبيرة. بالإضافة إلى ذلك، على الرغم من أن مستويات نقطة النهاية التعبير هي سهلة لتحديد معقول، يحتاج إلى المزيد من العمل الذي يتعين القيام به في فهم الديناميات الجوهرية لنظام خالية من الخلايا. ومن المعروف أن كلا competitio المواردن والموارد قيود يمكن أن تؤثر على ديناميات التعبير. على سبيل المثال، يمكن محدودة سيغما الذاتية 70 يؤدي إلى تشبع النظام مع زيادة إنتاج الحمض النووي قالب ملف تعريف التعبير مماثلة لتلك التي النوكليوتيدات أو استنزاف الأحماض الأمينية. 9،27 ومع ذلك، وديناميات لا يجب أن تكون مفهومة تماما للاستفادة من النظام. لزيادة نقية من الغلة، ويمكن أن يتم من خلال النهج الأمثل آلة التعلم. 28 أسئلة من التنافس على الموارد والحد يمكن معالجتها من خلال نماذج رياضية التحقق باستخدام البيانات التجريبية. هو الأمثل لبروتوكول المعروضة هنا للحصول على سلالة BL21-Rosetta2، ولكن هو للتعميم إلى أخرى E. سلالات القولونية. تعديلات في BL21-Rosetta2، مثل إزالة الجين الترميز خط الطول البروتيني وإضافة ترميز الجينات tRNAs نادرة، والسماح لإنتاج البروتين القصوى. حاولنا بروتوكول مع اثنين من سلالات استخراج أخرى - BL21 BL21 فقط وtrxA خروج المغلوب، ووجد د 50٪ أقل من البروتين العائد. نحن نفترض أن الغلات تنخفض بالمثل عند استخدام السلالات الأخرى. ، وقد أدت التغييرات الأخرى في المعلمات، مثل تبديل 2xYT متوسطة النمو لLB والمرق الغنية الأخرى في تناقص الغلة البروتين. نظم التعبير خالية من الخلايا باستخدام الداخلية والخارجية على حد سواء آلات النسخ الترجمة وآليات التنظيم لها تطبيقات واسعة في كل من البروتين والأيض والتعبير في البيولوجيا التركيبية. 3،29 بدلا من أن يقتصر على دوائر التنظيم T7، يمكن للمرء أن يتصور إنتاج الجزيئات الحيوية المعقدة في وضع المستخدم السيطرة عليها باستخدام مزيج من الأم E. المروجين القولونية وآليات النسخ وتنظيم الموردة خارجيا. دون قيود من انقسام الخلايا والتمثيل الغذائي، وتقلب في الدوائر الاصطناعية مثل repressilator أو في التمثيل الغذائي المهندسة مسارات مثل تلك التي تنتج مادة الأرتيميسينين يمكن تخفيض أو مفهومة على نحو أفضل. 30،31 نحن هفه تستخدم هذه المزايا لتنفيذ مفاتيح الوراثية، وكذلك لفهم سيغما عامل تنحية يمكن 9،32 هذه التكنولوجيا أيضا تشكل العمود الفقري لل&quot;الحد الأدنى&quot; أو خلايا &quot;مصطنعة&quot; - صغيرة، وتتميز بشكل جيد وحدات المجسدة الاكتفاء الذاتي من استخراج. 33،34 في نهاية المطاف، ونحن نتوقع الاستخدامات الفوري لهذا النظام حرية التعبير الخلايا الذاتية كبيئة النماذج للبيولوجيا الاصطناعية. الملقب ب &quot;TX-TL اللوح الجزيئية البيولوجية،&quot; يوفر نظام حرية التعبير خلايا بيئة يمكن السيطرة عليها حيث الدوائر الاصطناعية المخصصة في نهاية المطاف لفي الجسم الحي التعبير يمكن أن تخضع لجولات من النماذج - دورات الاختبار على البلازميد الأساسية، الخطي، أو الحمض النووي synthetized كيميائيا، يليه عن طريق التحليل والتعديل السريع. جولات النماذج يمكن بمساعدة النماذج الرياضية التنبؤية التي يجري تطويرها حاليا. عن طريق إزالة الاستنساخ والتلاعب في الجسم الحي للدوائر غير نهائية، ونحن نتوقع إنجيدورة مرات neering أن تنخفض إلى 1-3 أيام بدلا من معيار الأسابيع الحالية.

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

بدأت التكنولوجيا التعبير خالية من الخلايا في 1950s باعتبارها متعدية بحتة، والنهوض بعد سنوات لتشمل آليات النسخ الترجمة يقترن باستخدام الحمض النووي البكتيريا T7. 11،12 ومنذ ذلك الحين، بذلت جهود عديدة لتحسين إنشاء خلية استخراج الخام (أو E وتشمل. استخراج القولونية S30). 13،14 هذه التحسينات إطالة تخليق البروتين خالية من الخلايا من خلال تجديد ATP أو تعديلات السلالة، وتقليل الوقت والتكلفة بروتوكول. موجودة 15-17 نظم التعبير خالية من الخلايا البديلة التي أعيد استخدام مكونات بدلا من النفط الخام وقد وضعت استخراج خلية للتعبير. 5 كلا استخراج خلية الخام وأساليب إعادة للاستخدام التجاري. مع ظهور علم الأحياء الاصطناعية، وهناك حاجة متزايدة لمنصة تتميز جيدا لاختبار والتعبير عن وحدات ودوائر البيولوجية هندسيا. 18،19 يجب أن تكون هذه المنصةتنوعا، يتميز بشكل جيد، وسهلة التعامل معها، وتركز على مكونات الموفر من قبل المستخدم. على الرغم من كونها وضعت نصف قرن في وقت سابق، وأنظمة خالية من الخلايا استنادا إلى E. القولونية مشاركة في جوهرها هذه المتطلبات، كما هي مبسطة في التمثيل المختبر من العمليات الخلوية دون تعقيد النمو والأيض. بالإضافة إلى ذلك، كل من المعرفة التأسيسية من العمل في الجسم الحي على E. ينطبق القولونية E. بسهولة ل نظم خالية من الخلايا القولونية. على الرغم من أن أنظمة التعبير خالية من الخلايا يمكن أن تكون لها تطبيقات في مجال البيولوجيا الاصطناعية، وحتى الآن الهدف من معظم النظم التعبير خالية من الخلايا كان تعظيم العائد من البروتين والأيض. ويتم إنجاز هذا باستخدام البكتيريا T7 النسخ من تسلسل يقودها المروجين T7 20 على الرغم من أن التعبير هو كفاءة وقوية، وهذه النظم تخدم غرضا درجة عالية من التخصص. طرق تنظيم خلية محدودة، ويجب أن تكون قوالب الحمض النووي المستهدفصياغتها لتشمل المروجين T7، وبعض متواليات مثل المجمعات الريباسي لا يمكن نسخها وتجميعها. 21،22 نظم التعبير خالية من الخلايا الموجودة غير قادرة على الحفاظ على عوائد عالية مع الحفاظ على الآليات التنظيمية الذاتية، وبراعة اللازمة للبيولوجيا الاصطناعية. قمنا بتطوير E. الذاتية القولونية خالية من الخلايا نظام التعبير الذي يحافظ على كفاءة البروتين التعبير يتبين من الأنظمة السابقة ولكنها تضيف براعة إضافية من خلال السماح التعبير والتنظيم على أساس كل من (T7 أو غيرها) آليات الداخلية والخارجية. بروتوكول الموصوفة هنا يستند أصلا على Kigawa وآخرون (2004) وليو وآخرون (2005)، ولكن لديه تعديلات كبيرة. فإنه يستخدم المغنيسيوم وK-الغلوتامات على المغنيسيوم وK-خلات لزيادة الكفاءة، ويزيل 2 المركابتويثانول، وlyses الخلايا باستخدام حبة الخافق. يتم اختيار 17،23،24 الخرزة ضرب على التجانس،على أساس أساليب الضغط، أو صوتنة بسبب انخفاض تكلفة وعائدات مماثلة لأنظمة المتنافسة. يستخدم حمض 23 3 phosphoglyceric (3-PGA) كمصدر للطاقة كما وجد أن تعطي غلة البروتين متفوقة بالمقارنة مع فوسفات الكرياتين و الفسفوإينول. 4،25 نظامنا يمكن ان تنتج ما يصل الى 0.75 ملغ / مل من البروتين مراسل باستخدام إما المروج القائم على sigma70 مع مشغلي امدا فج أو المروج T7 يحركها، مماثلة لغلة من الأنظمة التجارية الأخرى. 4،6 خمسة أيام هناك حاجة لانتاج جميع الكواشف اللازمة (الشكل 1). وعلاوة على ذلك، فإنه يوفر خفض التكاليف 98٪ بالمقارنة مع أنظمة مماثلة خالية من الخلايا التجارية - التكاليف المادية هي 0،11 $ لكل 10 ميكرولتر رد فعل، والتي ترتفع إلى 0،26 $ مع تضمين العمل (الشكل 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

ويمكن لنظم التعبير خالية من الخلايا مثالية نظريا محاكاة بيئة الخلوية في الجسم الحي في رقابة في منصة المختبر. 1 وهذا مفيد للتعبير عن البروتينات ودوائر الوراثية في الطريقة التي تسيطر عليها فضلا عن توفير بيئة النماذج الأولية للبيولوجيا الاصطناعية. 2،3 لتحقيق هذا الهدف الأخير، ونظم التعبير خالية من الخلايا التي تحافظ على آليات النسخ الترجمة الذاتية كولاي هي قادرة أن تعكس بشكل أكثر دقة في الجسم الحي ديناميات الخلوية من تلك القائمة على الحمض النووي الريبي بوليميراز T7 النسخ. نحن تصف إعداد وتنفيذ وكفاءة الذاتية E. القولونية يستند النسخ الترجمة (TX-TL) خالية من الخلايا نظام التعبير التي يمكن أن تنتج كميات من البروتين كما تعادل النظم القائمة T7-في خفض التكاليف 98٪ إلى النظم التجارية مشابهة. 4،5 إعداد مخازن واستخراج خلية الخام وصفها، وكذلك تنفيذا لثلاثة أنبوب TX-TL رد الفعل. بروتوكول كامل يستغرق خمسة أيام لإعداد وينتج ما يكفي من المواد لمدة تصل إلى 3000 ردود فعل واحد في التحضير واحد. مرة واحدة على استعداد، ويأخذ كل رد فعل أقل من 8 ساعة من الإعداد لجمع البيانات وتحليلها. آليات التنظيم والنسخ الخارجية إلى E. القولونية، مثل repressors أمريكا اللاتينية والكاريبي / تيت وT7 RNA البلمرة، يمكن أن تستكمل. 6 الخصائص الذاتية، مثل مرنا ومعدلات تدهور الحمض النووي، كما يمكن تعديلها. 7 وقد تجلى هذا النظام حرية التعبير خلايا TX-TL لواسع تتوفر الجمعية على نطاق الدائرة، واستكشاف الظواهر البيولوجية، والتعبير عن البروتينات في إطار كل من المروجين T7 والذاتية. 6،8 النماذج الرياضية المصاحبة. 9،10 النظام الناتجة لها تطبيقات فريدة من نوعها في مجال البيولوجيا التركيبية وبيئة النماذج، أو &quot;TX- TL الجزيئية البيولوجية اللوح &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

يحدد هذا البروتوكول لمدة خمسة أيام كل الخطوات والمعدات والبرمجيات اللازمة تكميلية لخلق وتشغيل فعال الذاتية القولونية يستند TX-TL نظام التعبير خالية من الخلايا من الصفر. مع الكواشف، وبروتوكول يأخذ 8 ساعات أو أقل لإعداد رد فعل، وجمع، ومعالجة البيانات.

بروتوكولات لتنفيذ و<em&gt; كولاي</em&gt; وبناء خلية الحرة TX-TL نظام التعبير عن علم الأحياء الاصطناعية

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.  يحدد هذا البروتوكول لمدة خمسة أيام كل الخطوات والمعدات والبرمجيات اللازمة تكميلية لخلق وتشغيل فعال الذاتية القولونية يستند TX-TL نظام التعبير خالية من الخلايا من الصفر. مع الكواشف، وبروتوكول يأخذ 8 ساعات أو أقل لإعداد رد فعل، وجمع، ومع?...

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.

على الفحص لقياس نشاط القولونية محرض يسين 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.

بروتوكولات اختيار الطفرات وظيفية لتطور المخرج من البروتينات في E. القولونية</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.

ارتفاع المناولة فحص النشاط Endoglucanase الفطرية في القولونية</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.

رسم خرائط الشبكات الوظيفية البكتيرية والممرات في<em&gt; كولاي</em&gt; استخدام المصفوفات الوراثية الاصطناعية

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.

بروتوكول السريع لإعداد Electrocompetent<em&gt; كولاي</em&gt; و<em&gt; ضمة الكوليرا</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

الكشف عن لايف<em&gt; كولاي</em&gt; O157: H7 من قبل خلايا 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.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</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.

فوائد متعددة الأوجه من البروتين المشارك في التعبير<em&gt; الإشريكية القولونية</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.

نظم هيلا خلية استنادا حرية التعبير للتعبير عن<em&gt; المتصورة</em&gt; المقيرع البروتينات

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.