المؤلفون ممتنون للدكتور توماس Rademacher عن توفير pPAM محطة ناقلات التعبير وإبراهيم أميدي للزراعة نباتات التبغ المستخدمة في هذه الدراسة. نود أن نشكر الدكتور ريتشارد م. Twyman لمساعدته في تحرير المخطوطة. كان في جزء منه تمويل هذا العمل من قبل مجلس البحوث الأوروبي المتقدم غرانت &quot;المستقبل-فارما&quot;، اقتراح عدد 269110 وفراونهوفر Zukunftsstiftung (مؤسسة فراونهوفر المستقبل).

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وقد رعت رسوم النشر بصورة جزئية من قبل الشركات Statease، وشركة (الولايات المتحدة الأمريكية) وStatcon (ألمانيا)، والتي لم تشارك في المشاركة في إعداد المخطوط أو مسؤولة عن أي من محتوياته.

كل تجربة يتطلب تخطيطا دقيقا لأن الموارد غالبا ما تكون نادرة وباهظة الثمن. هذا صحيح بصفة خاصة للاستراتيجيات وزارة الطاقة هذا بسبب أخطاء أثناء مرحلة التخطيط (على سبيل المثال اختيار نموذج القاعدة التي لا تغطي جميع التفاعلات عامل هام) يمكن أن يقلل بشكل كبير من القوة...

إنتاج البروتينات الصيدلانية البيولوجية في النباتات هو مفيد لأن النباتات هي غير مكلفة لتنمو، ومنصة يمكن زيادتها فقط من خلال زراعة المزيد من النباتات، والكائنات الممرضة للإنسان غير قادر على تكرار 1،2. استراتيجيات التعبير عابرة تستند على سبيل المثال على التسلل من الأوراق مع الأجرعية المورمة يوفر فوائد إضافية لأن يتم تقليل الوقت بين نقطة التسليم الحمض النووي وتسليم منتج تنقيته من سنة إلى أقل من 2 شهور 3. يستخدم التعبير عابرة أيضا لتحليل وظيفي، على سبيل المثال لاختبار الجينات لقدرتها على استكمال الخسارة من وظيفة المسوخ أو للتحقيق تفاعلات البروتين 4-6. ومع ذلك، ومستويات التعبير عابرة تميل إلى إظهار قدر أكبر من التباين دفعة إلى دفعة من مستويات التعبير في النباتات المعدلة وراثيا 7-9. وهذا يقلل من احتمال أن عمليات التصنيع الصيدلانية البيولوجية على أساس عابر واي التعبيرليرة لبنانية تتم الموافقة عليها في سياق الممارسات التصنيعية الجيدة (GMP) لاستنساخ هو سمة الجودة الحرجة، ويخضع لتقييم المخاطر 10. يمكن لمثل الاختلاف أيضا قناع أي التفاعلات التي تنوي الباحثين للتحقيق. لذلك، شرعنا في تحديد العوامل الرئيسية التي تؤثر على مستويات التعبير عابرة في النباتات وبناء ذات جودة عالية نموذج تنبؤي الكمي. وغالبا ما يستخدم (OFAT) نهج عامل واحد في واحد في وقت لتوصيف تأثير (أثر) من بعض المعلمات (العوامل) على النتيجة (الاستجابة) من تجربة 11. ولكن هذا هو الأمثل لأن الاختبارات الفردية (يدير) أثناء التحقيق (التجربة) سيتم محاذاة مثل اللؤلؤ على سلسلة عبر المنطقة المحتملة امتدت من العوامل التي يتم اختبارها (تصميم الفضاء). تغطية تصميم الفضاء، وبالتالي درجة المعلومات المستقاة من التجربة هومنخفضة، كما هو مبين في الشكل 1A 12. علاوة على ذلك، يمكن أن الترابط بين عوامل مختلفة (عامل التفاعلات) لا تزال مخبأة مما أدى إلى نماذج الفقراء و / أو التنبؤ أوبتيما كاذبة، كما هو مبين في الشكل 1B 13. السلبيات المذكورة أعلاه يمكن تجنبها باستخدام تصميم التجارب (وزارة الطاقة) في النهج الذي تنتشر تشغيل من تجربة أكثر بالتساوي في جميع أنحاء تصميم الفضاء، وهذا يعني أن أكثر من عامل واحد وتتنوع بين اثنان أشواط 14. هناك تصاميم المتخصصة لمخاليط، وفحص العوامل (تصاميم مضروب) والكميات من آثار عامل على الردود (أساليب السطح ردا على ذلك، RSM ق) 15. علاوة على ذلك، RSMs يمكن أن تتحقق عن تصاميم المركزية مركب ولكن يمكن أيضا أن يتحقق على نحو فعال باستخدام البرمجيات المتخصصة التي يمكن أن تطبق معايير مختلفة لاختيار أشواط. على سبيل المثال، ما يسمى D-optimalitسوف ذ معيار تحديد أشواط ذلك لتقليل الخطأ في معاملات النموذج الناتج، في حين أن المعيار IV-المثالية يختار أشواط أن تحقيق أدنى التنبؤ التباين في جميع أنحاء تصميم الفضاء 15،16. في RSM وصفنا هنا يسمح الكميات الدقيقة لبروتين تعبير عابر في النباتات، ولكن يمكن بسهولة أن يتم نقلها إلى أي نظام ينطوي عدة (~ 5-8) العوامل الرقمية (مثل درجة الحرارة والوقت والتركيز) وعدد قليل (~ 2 - 4) العوامل قاطعة (مثل المروج، لون) الذي وصف الآلية غير متوفرة أو معقدة للغاية في تصميم نموذج. النهج زارة الطاقة نشأت في العلوم الزراعية ولكن قد انتشر إلى مناطق أخرى لأنها قابلة للتحويل إلى أي الحالة التي يكون فيها من المفيد للحد من عدد من يدير اللازمة للحصول على بيانات موثوقة وتوليد نماذج وصفية لعمليات معقدة. وهذا بدوره أدى إلى إدراج زارة الطاقة في &quot;إرشادات لالصناعة، Q8 (R2) تطوير ادوية &quot;الصادرة عن المؤتمر الدولي لمواءمة المتطلبات التقنية لتسجيل المستحضرات الصيدلانية للاستخدام البشري (ICH) 17. يستخدم على نطاق واسع الآن زارة الطاقة في مجال البحث العلمي والصناعة 18. ومع ذلك، يجب توخي الحذر خلال تخطيط وتنفيذ التجربة لاختيار درجة متعدد الحدود غير لائق لنموذج متعدد الانحدار الخطي (نموذج القاعدة) يمكن أن يعرض حاجة لأشواط إضافية لنموذج جميع الآثار عامل بشكل صحيح. وعلاوة على ذلك، تالفة أو مفقودة توليد نماذج البيانات غير صحيحة وخاطئة التوقعات، وربما حتى منع أي محاولة بناء نموذج كما هو موضح في البروتوكول والمناقشة أقسام 18. في قسم البروتوكول، ونحن سوف تحدد في البداية من الخطوات الأكثر أهمية التخطيط للتجربة تستند RSM-ثم شرح التصميم على أساس وزارة الطاقة برامج DesignExpert V8.1، ولكن تصاميم مماثلة يمكن أن يبنى مع غيرها من البرمجيات includiنانوغرام أحزاب اللقاء المشترك، Modde، وSTATISTICA. يتم اتباع الإجراءات التجريبية حسب تعليمات لتحليل البيانات والتقييم. <img alt="الشكل 1" fo:content-width="5in" fo:src="/files/ftp_upload/51216/51216fig1highres.jpg" src="/files/ftp_upload/51216/51216fig1.jpg" /> الشكل 1. مقارنة OFAT وزارة الطاقة. A. تباين متتابعة من عامل واحد في وقت واحد (OFAT) في تجربة (الدوائر الأسود والأحمر والأزرق) يحقق التغطية المنخفضة من تصميم الفضاء (المناطق التي تحاك). في المقابل، فإن الاختلاف من أكثر من عامل واحد في وقت واحد باستخدام تصميم التجارب (وزارة الطاقة) استراتيجية (الدوائر الخضراء) يعزز التغطية وبالتالي دقة النماذج الناتجة عن ذلك. ب. التغطية المتحيزة تصميم الفضاء يعني أن التجارب OFAT (الدوائر السوداء) يمكن أن تفشل أيضا لتحديد مناطق التشغيل الأمثل (الحمراء)، والتنبؤ الحلول دون المستوى الأمثل (دائرة سوداء كبيرة)، في حين أن وزارة الطاقة strategiوفاق (النجوم السوداء) هم أكثر عرضة لتحديد الظروف الأفضل (نجمة سوداء كبيرة).

<table border="0" cellpadding="0" cellspacing="0" width="946">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td style="width:272px;">DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tryptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8952.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2363.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ampicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">K029.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agar-Agar</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">5210.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Escherichia coli K12 DH5a</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">18263-012</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">pPAM</td>
			<td style="width:195px;">GenBank</td>
			<td style="width:119px;">AY027531</td>
			<td>Cloning/expression vector;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Plasmid&#160;</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740588.250</td>
			<td>Plasmid DNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NucleoSpin Gel and PCR Clean-up</td>
			<td style="width:195px;">MACHEREY-NAGEL GmbH</td>
			<td style="width:119px;">740609.250</td>
			<td>Plasmid DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NanoDrop 2000</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">n.a.</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NcoI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3193L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EcoRI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R3101L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">AscI</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">R0558L</td>
			<td>Restrictionendonuclease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NEB 4</td>
			<td style="width:195px;">New England Biolabs Inc.</td>
			<td style="width:119px;">B7004S</td>
			<td>Restrictionendonuclease buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TRIS</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4855.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium tetraborate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4403.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8040.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6352.4</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bromophenol blue</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A512.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Xylene cyanol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">A513.1</td>
			<td>Color indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glycerol</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">7530.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Mini-Sub Cell GT Cell</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">170-4406</td>
			<td>Gel electrophoresis chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agrobacterium tumefaciens strain GV3101:pMP90RK</td>
			<td style="width:195px;">DSMZ</td>
			<td style="width:119px;">12365</td>
			<td>Microorganism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Electroporator 2510</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">4307000.658</td>
			<td>Electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Beef extract</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">X975.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Peptone</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">2365.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sucrose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4621.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magnesium sulfate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">0261.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Carbenicillin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6344.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Kanamycin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">T832.3</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Rifampicin</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4163.2</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FWD primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCT CAG GAA GAG CAA TAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">REV primer</td>
			<td style="width:195px;">Eurofins MWG Operon</td>
			<td style="width:119px;">n.a.</td>
			<td>CCA AAG CGA GTA CAC AAC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2720 Thermal cycler</td>
			<td style="width:195px;">Applied Biosystems</td>
			<td style="width:119px;">4359659</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">RNAfold webserver</td>
			<td style="width:195px;">University of Vienna</td>
			<td style="width:119px;">n.a.</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">n.a.</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Omnifix-F Solo</td>
			<td style="width:195px;">B. Braun</td>
			<td style="width:119px;">6064204</td>
			<td>Syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Murashige and Skoog salts</td>
			<td style="width:195px;">Duchefa</td>
			<td style="width:119px;">M 0222.0010</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glucose</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">6780.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Acetosyringone</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">D134406-5G</td>
			<td>Phytohormon analogon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">&#160;BioPhotometer plus</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">&#160;6132 000.008</td>
			<td>Photometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">n.a.</td>
			<td>SPR device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Protein A</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">10-1006</td>
			<td>Antibody binding protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tween-20</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9127.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2G12 antibody</td>
			<td style="width:195px;">Polymun</td>
			<td style="width:119px;">AB002</td>
			<td>Reference antibody</td>
		</tr>
	</tbody>
</table>

characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study

توفر النباتات فوائد متعددة لإنتاج المستحضرات الصيدلانية البيولوجية بما في ذلك تكاليف منخفضة، والتدرجية، والسلامة. يقدم التعبير عابرة ميزة إضافية للتنمية قصيرة والأوقات الإنتاج، ولكن يمكن لمستويات التعبير تتفاوت تفاوتا كبيرا بين دفعات بالتالي مما أدى إلى مخاوف تنظيمية في سياق الممارسات التصنيعية الجيدة. استخدمنا تصميم التجارب (وزارة الطاقة) نهج لتحديد تأثير العوامل الرئيسية مثل العناصر التنظيمية في بناء التعبير ونمو النبات والمعلمات التنمية، وشروط الحضانة خلال التعبير، على تنوع التعبير بين دفعات. اختبرنا النباتات تعبر عن مكافحة فيروس نقص المناعة البشرية الأضداد وحيدة النسيلة نموذج (2G12) والبروتين علامة فلوري (DsRed). نناقش الأساس المنطقي لاختيار خصائص معينة من النموذج وتحديد حدودها المحتملة. يمكن بسهولة نقل النهج العام إلى مشاكل أخرى لأن مبادئ نموذج لإعادة على نطاق واسع للتطبيق: القائم على المعرفة اختيار المعلمة، والحد من التعقيد عن طريق تقسيم المشكلة إلى وحدات أصغر الأولية، وإعداد برامج موجهة لمجموعات التجربة الأمثل وتصميم خطوة حكيمة تكبير. وبالتالي، فإن منهجية مفيد لوصف البروتين التعبير في النباتات ولكن أيضا للتحقيق في الأنظمة المعقدة الأخرى التي تفتقر إلى وصف الآلية ليس فقط. المعادلات التنبؤية واصفا الترابط بين المعلمات يمكن استخدامها لإنشاء نماذج الآلي لأنظمة معقدة أخرى.

نموذج وصفي لتراكم DsRed خلال التعبير عابرة استخدام منشطات و5&#39;UTRs مختلفة وقد استخدم مضان DsRed في مقتطفات ورقة تشير إلى مستوى التعبير من البروتين المؤتلف وبالتالي تم استخدامه باعتباره استجابة في استراتيجية وزارة الطاقة. كان...

Watch this Scientific Journal Video about Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study at JoVE.com

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

نحن تصف تصميم التجارب النهج التي يمكن استخدامها لتحديد ونمذجة تأثير العناصر التحوير التنظيمية والنمو والتنمية المعلمات النبات، وشروط الحضانة على التعبير عابرة من الاجسام المضادة والبروتينات مراسل في النباتات.

توصيف النظم المعقدة باستخدام تصميم التجارب النهج: عابر التعبير البروتين في التبغ كدراسة حالة

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

characterization-complex-systems-using-design-experiments-approach

Research

JoVE Journal

Bioengineering

16.4K Views.  RWTH Aachen University.  نحن تصف تصميم التجارب النهج التي يمكن استخدامها لتحديد ونمذجة تأثير العناصر التحوير التنظيمية والنمو والتنمية المعلمات النبات، وشروط الحضانة على التعبير عابرة من الاجسام المضادة والبروتينات مراسل في النباتات. 

Video: Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

عالية الإنتاجية مولد بروتين تعبير باستخدام منصة ميكروفلويديك

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

اختبار Microtensile التي تسيطر عليها بيئيا للميكانيكيا على التكيف nanocomposites والبوليمرات لل<em&gt; خارج الحي</em&gt; توصيف

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

A منصة الآلي تنوعا للتجارب تحفيز الخلية الصغيرة الحجم

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

رصد في الوقت الحقيقي من بين الخلايا الصفراء حمض حيوية باستخدام حمض الصفراء الاستشعار القائمة على الحنق ترميز وراثيا

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

كشف الذيفان في تركيبات النانو باستخدام فحوصات ليستي (ال) أموبوسيتي ليمولوس

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

التعبير عابر في البنجر الأحمر لقاح مرشح الصيدلانية البيولوجية لمرض السكري نوع 1

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

منصة ميكروفلويدريك متعددة الطبقات لتوصيل التعبير الجيني الخالي من الخلايا لفترات طويلة

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

في فيسيكولو التوليف من السلائف غشاء الببتيد لنمو الحويصلة المستقلة

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

تعريف عالي الإنتاجية للمقاومة لـ Pseudomonas syringae pv. الطماطم في الطماطم باستخدام الشتلات الفيضانات أساي

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

التشريح المجهري للطبقة من وريقات الصمام ثلاثي الشرف للتوصيف الميكانيكي ثنائي المحور والتكميم الهيكلي الدقيق

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...