أرقام 1، 2، 3، 4، 6، 8 و الجداول 2 و 3 و 10 و 15 و 16 يتم تعديلها من الرقم 29. وأيد هذا العمل من قبل مؤسسة العلوم الوطنية (NSF-منح CCF 1117143 وNSF-CCF 1162141 لGS). وأيد Y.-JC التي كتبها الزمالات الحكومة التايوانية. وأيد SDR من قبل البرنامج الوطني للعلوم مؤسسة العليا للبحوث زمالة (GRFP).

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The authors declare no competing financial interests.

<html> وتصف هذه الورقة طريقة لاشتقاق بوابات ndsDNA من DNA البلازميد نقية للغاية. وعلاوة على ذلك، يتم تقديم بروتوكول للتميز أداء البوابة باستخدام فحص حركية مضان. وتبين بيانات التجارب أن النظام المستمدة من البلازميد يتفوق نظيرتها الاصطناعية حتى لو تم تجميعها في نظام الاصطناعية من خيوط تنقيته باستخدام هلام بولي أكريلاميد الكهربائي (PAGE). على الأرجح، ويرجع ذلك في المقام الأول إلى نقاء عالية جدا من الحمض النووي البيولوجي في تحسين أداء بوابات المستمدة من البلازميد. يحتوي على الحمض النووي الاصطناعي مجموعة متنوعة من الأخطاء، في الحذف معينة التي تؤدي إلى أليغنوكليوتيد] من طول هذه المنتجات الجانبية ن 1، وعادة ما يتم إزالة تماما في PAGE أو عالية الأداء اللوني السائل (HPLC) إجراءات التنقية. وقد لوحظت تحسينات مماثلة لتلك التي ذكرت هنا أيضا في دراسة سابقة من مكبر للصوت دبوس المحفزة التي تستخدم DNA المستمدة من مصادر بيولوجية 21. الحمار = &quot;jove_content&quot;&gt; ومع ذلك، وحتى استخدام البوابات المستمدة من البلازميد لا يمكن القضاء على الأخطاء في أداء البوابة، التي هناك سببين على الأقل تماما: أولا الإفراط في الهضم أو عدم وجود قطع الدقة يمكن أن يؤدي إلى البوابات مع عدد كبير جدا النكات أو النكات في مواضع خاطئة. في كلتا الحالتين، هم أكثر عرضة للمشاركة في ردود فعل غير مرغوب فيها البوابات. قد خففت هذه المشاكل عن طريق الاستفادة المثلى من كمية الإنزيم المستخدم (انظر الشكل 4). ثانيا، في هذه التجارب، وكانت معظم المدخلات وخيوط المساعدة DNA الاصطناعية وتضمنت الحذف وطفرات الآن. من حيث المبدأ، يمكن أيضا الحصول على كل مدخل واحد الذين تقطعت بهم السبل وخيوط المساعدة من DNA phagemid من خلال انزيم الهضم الخدش من الجينوم الفيروسي M13 المشفرة قبل 26. ولعل أداء الدائرة ويمكن زيادة تحسين استخدام ssDNA المستمدة من الجينوم البكتيري. في حين تم العثور على استخدام بوابات المستمدة من البلازميد لتحسين أداء الدوائر، تحليلالعصر التكلفة وتجهيز كشف أنه في حين أن إنتاج البوابات المستمدة من البلازميد هو أرخص قليلا (الجدول 15)، فإنه يأخذ 2-3 مرات أطول وقت المعالجة مقارنة مع التجمع وتنقية البوابات من oligos تصنيعه تجاريا (الجدول 16). التكاليف الأولية للبوابات المستمدة من البلازميد هي التوليف الجيني واستخدام أنزيمات التقييد. 300 pmole من بوابات (ما يكفي لمدة 15 ردود الفعل في 30 نانومتر)، وتبلغ التكلفة التقديرية للتاريخ أبواب ما يقرب من 170 $ و 200 $ للشوكة البوابات، والفرق في التكاليف كونها نتيجة لاستخدام الإنزيمات الخدش مختلفة. في المقابل، فإن التركيب الكيميائي للفروع لتغطية تكاليف البوابة نفسها حوالي 260 $ بما في ذلك رسوم تنقية PAGE. تكلفة الوقت الأولية للبوابات المستمدة من البلازميد هو في إجراء الاستنساخ، التي، تماما مثل الحمض النووي، يمكن الاستعانة بمصادر خارجية لشركة التوليف الجيني. ومع ذلك، مرة واحدة تجميعها، والبوابات المستمدة من البلازميد لديها ميزة أن البلازميدات المضيف يمكن بسهولة أن تتكرر لالثانية يمكن تخزينها في شكل أسهم الجلسرين البكتيرية. هذا يجعل من الممكن إعادة استخدام بوابات عدة مرات. نتطلع إلى الأمام، يمكن أن تحسن أداء بوابات المستمدة من البلازميد تمكين مجموعة أكبر بكثير من ديناميات السلوك من أثبتت تجريبيا حتى الآن مع DNA CRNs. على سبيل المثال، اقترح مؤخرا 47،48 العمل النظري الذي الأنماط المكانية ذاتية التنظيم على المستوى الكلي يمكن أن تتحقق مع CRNs DNA من خلال آلية نشر رد. الطريقة المعروضة هنا يوفر مسارا قابلا للتطبيق لبناء المكونات الجزيئية الكامنة لهذه المواد DNA-الزخرفة النفس. بالرغم من صعوبتها، فإن وضع الأشكال التضاريسية المستوى الكلي في طريقة برمجة لها آثار كبيرة في مجالات تتراوح من الأبحاث الحيوية في الطب التجديدي.

وقد أتاح إمكانية التنبؤ واتسون-كريك قاعدة الاقتران تكنولوجيا النانو DNA الديناميكي لتظهر وكأنها وسيلة للبرمجة لتصميم الأجهزة الجزيئية مع الخصائص الحيوية 1،2. على وجه الخصوص، ضفيرة DNA النزوح - للبرمجة، رد فعل التهجين تنافسية - وقد ثبت أن آلية قوية لهندسة النظم الحيوية DNA. في رد فعل النزوح حبلا DNA، وهو قليل النوكليوتيد واردة نزوح وملزمة من قبل حبلا &quot;الإخراج&quot; من شريك ملزم التكميلي. ردود الفعل هذه متعددة يمكن بالسلاسل معا في شلالات رد فعل متعددة الخطوات مع وجود درجة عالية من السيطرة على النظام وتوقيت رد الفعل الفردي الخطوات 3. وقد استخدمت DNA شلالات حبلا النزوح إلى إنشاء الدوائر الرقمية والتناظرية الجزيئية 4-7، النانو للتحويل 10/08، 15/11 المحركات الجزيئية مستقلة، ومكبرات الصوت الحفازة غير التساهمية 13،16-21. وعلاوة على ذلك، Dأجهزة NA باستخدام تفاعلات النزوح حبلا يمكن محاكاة ومصممة لتطبيقات متنوعة باستخدام بمساعدة الحاسوب تصميم البرمجيات 22-24. ، يقدم DNA تصنيعه كيميائيا حاليا كمادة رئيسية لتكنولوجيا النانو DNA. ومع ذلك، وأخطاء في عملية توليف الحمض النووي، وأليغنوكليوتيد الكمال الناتجة عن ذلك، ويعتقد أن للحد من أداء الأجهزة الحيوية DNA عن طريق التسبب في التفاعلات الجانبية الخاطئة. على سبيل المثال، وردود الفعل &quot;تسرب&quot; يمكن أن يؤدي إلى الإفراج عن النوكليوتيد الانتاج حتى في حالة عدم وجود رد فعل الزناد. هذه الآثار هي الأكثر وضوحا في شلالات رد فعل بالحفز فيها حتى الحد الأدنى من تسرب الأولي سوف يؤدي في نهاية المطاف في تفعيل كامل لل19،20 تتالي. على العكس من ذلك، غالبا ما تفشل ردود الفعل للوصول إلى المستوى المتوقع من تفعيل لبعض المكونات لا يؤدي حتى في وجود مدخلات المقصود 7،25. لجعل أداء DNA يعمل بنظام التشغيلالأجهزة النانوية مماثلة لنظرائهم القائم على البروتين البيولوجية، يتعين خفضت بشكل كبير هذه سائط الخطأ. البلازميدات البكتيرية أو DNA البيولوجية الأخرى يمكن أن تكون مصدرا رخيصا نسبيا من الحمض النووي نقية للغاية لتطبيقات تكنولوجيا النانو. يمكن توليد كميات كبيرة من الحمض النووي عن طريق تكرارها في البكتيريا وقدرات التدقيق الجوهرية للنظم المعيشة ضمان نقاء DNA الناتجة عن ذلك. في الواقع، وقد اعترفت عدة ورقات الأخيرة الفائدة المحتملة من DNA البيولوجي لتطبيقات تكنولوجيا النانو 21،26-28. ومع ذلك، فإن طبيعة المزدوج تقطعت بهم السبل تماما DNA البلازميد حظرت حتى الآن استخدامه كمادة لصنع أجهزة DNA الحيوية، والتي تتكون عادة من [أليغنوكليوتيد متعددة وتحتوي على كل المجالات المزدوج تقطعت بهم السبل واحدة الذين تقطعت بهم السبل. في ورقة حديثة 29 تم تناول هذه المسألة، وهيكل جديد بوابة DNA التي تتكون أساسا من الحمض النووي المزدوج تقطعت بهم السبل رصدت (ndsDNA) كان إدخالد. الأهم من ذلك، أنظمة البوابات ndsDNA يمكن تصميم أن ندرك ديناميات المحدد من قبل أي شبكة التفاعل الكيميائي الرسمية (CRN) 29. وبالتالي يمكن استخدام بوابات ndsDNA، من حيث المبدأ، لإنشاء الأنظمة الديناميكية التي تظهر التذبذبات والفوضى، bistability والذاكرة، والمنطق منطقي أو السلوكيات حسابي 30-38. على سبيل المثال، المرجع 29 أظهرت رد فعل CRN ثلاث سنوات التي وفرت تنفيذ الجزيئي للبروتوكول &quot;الإجماع&quot;، وهو نوع من توزيع خوارزمية الحوسبة 29،39،40. تظاهر هذا العمل لأول مرة استخدام الرواية لالشكلية CRN بأنها &quot;لغة البرمجة&quot; لتوليف بسرعة الأنظمة الجزيئية وظيفية (الشكل 1A). هنا، يتم توفير بروتوكول مفصلة لاشتقاق بوابات ndsDNA من DNA البلازميد. الأول هو إعادة النظر في عملية التصميم التسلسل. ثم يلي شرح لكيفية [أليغنوكليوتيد الاصطناعية التي تحتوي علىيتم استنساخ تسلسل البوابة إلى البلازميدات وتسلسل التحقق وتضخيمها عبر ثقافة البكتيرية. بعد ذلك، فإنه يظهر كيف يمكن أن تستمد ndsDNA البوابات من البلازميدات عن طريق تجهيز الأنزيمي (انظر الشكل 2). وأخيرا، الخطوط العريضة لطريقة لاختبار السلوك البوابة باستخدام حركية مضان فحوصات. آلية رد الفعل وكمثال على ذلك، يركز البروتوكول على الحفاز تفاعل كيميائي A + B-&gt; B + C. الأنواع A، B، C و(&quot;إشارات&quot;، الشكل 1B) كل تتوافق مع احد الذين تقطعت بهم السبل مختلف جزيء DNA. تسلسل هذه الجزيئات هي مستقلة تماما وخيوط لا تتفاعل مع بعضها البعض مباشرة. تسلسل جميع الاشارات واثنين من المجالات الوظيفية المختلفة، أي المتتالية والتي تعمل معا في التفاعلات النزوح حبلا: 1) مجال موطئ قدم قصيرة (علامات تا، السل، TC) التي يتم استخدامها لبدء حبلا النزوح صeaction، و2) مجال طويلة (تسميات أ، ب، ج) أن يحدد هوية إشارة. وبوساطة التفاعلات بين مسارات الإشارات التي رصدت DNA (ndsDNA) المجمعات بوابة المزدوج تقطعت بهم السبل (وتسمى إنضم AB وشوكة قبل الميلاد)، ونوع واحد الذين تقطعت بهم السبل المساعدة (&lt;tr ص&gt;، &lt;ب tr&gt; &lt;ج السل&gt; و &lt;ط ح &gt;). يتم تنفيذ رد فعل رسمي A + B-&gt; B + C من خلال سلسلة من حبلا النزوح خطوات التفاعل، حيث تكشف كل خطوة رد فعل على موطئ قدم لرد فعل لاحق (الشكل 1B). في هذا المثال، إشارات A و B في البداية الحرة في حل بينما إشارة C منضما إلى بوابة شوكة. في نهاية التفاعل B و C في الحل. بشكل أعم، الإشارات التي لا بد أن بوابة غير نشطة بينما الإشارات التي هي حرة في حل نشطة، وهذا يعني أنها يمكن أن تشارك في رد فعل حبلا التشردمدخلا. ويتبع مسار وقت رد الفعل باستخدام استراتيجية مراسل فلوري (الشكل 1C). في الأعمال السابقة 29، وقد تبين أن هذه الآلية رد فعل يدرك ليس فقط رياضيات الكيمياء الصحيح ولكن أيضا حركية التفاعل الهدف.

<table border="0">
	<tbody>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>NEB</td>
			<td>M0531S</td>
			<td></td>
		</tr>
		<tr>
			<td>PvuII-HF</td>
			<td>NEB</td>
			<td>R3151L</td>
			<td></td>
		</tr>
		<tr>
			<td>PstI-HF</td>
			<td>NEB</td>
			<td>R3140S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth, Modified</td>
			<td>SIGMA-ALDRICH</td>
			<td>T0918-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Hispeed Maxi-prep Kit</td>
			<td>QIAGEN</td>
			<td>12662</td>
			<td></td>
		</tr>
		<tr>
			<td>Nb.BsrDI</td>
			<td>NEB</td>
			<td>R0648L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nt.BstNBI</td>
			<td>NEB</td>
			<td>R0607L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-stranded Genomic Blocks</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horiba Jobin-Yvon Spex Fluorolog-3 Fluorimeter</td>
			<td>Horiba/Jobin Yvon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Quartz Cells&#160;</td>
			<td>Starna</td>
			<td>23-5.45-S0G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Backbones</td>
			<td>BioBrick</td>
			<td>E0240-pSB1A2</td>
			<td>High copy number plasmid with Ampicillin resistance. Sequence can be found from http://parts.igem.org</td>
		</tr>
	</tbody>
</table>

plasmid-derived dna strand displacement gates for implementing chemical reaction networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA ستراند المهجرين غيتس لشبكات التفاعل التنفيذية الكيميائية المشتقة من البلازميد

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

plasmid-derived-dna-strand-displacement-gates-for-implementing

Universidad de Cartagena UDEC

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JoVE Journal

Biology

14.3K Views.  University of Washington. This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. 

Video: Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

تحليل مزدوج الجديلة إصلاح الحمض النووي (DSB) فاصل في خلايا الثدييات

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

كيفية تسجيل والثقافة والحث على الشبكات العصبية الصغيرة مسرى صالحة (الاتفاقات)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

الهندسة الحيوية شبكات الاوعية الدموية الدقيقة الإنسان في الفئران العوز المناعي

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

تكرارية الأمثل من دوبلكسات DNA لبلورة-DNA من SeqA مجمعات

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

تحديد البكتيريا عملية الأيض نشطة في الوتر من اختصاصي<em&gt; ورق القطن littoralis</em&gt; عن طريق الحمض النووي النظائر مستقرة جس طريق<sup&gt; 13</sup&gt; C-الجلوكوز

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

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

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

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

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

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

التصوير في الوقت الحقيقي من واحدة المهندسة رنا النصوص في الخلايا الحية عن طريق Ratiometric ثنائي الجزيء منارات

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

انخفاض الجاذبية المظاهرات الأجهزة البيئة في نموذج المنمنمة تدفق عداد الكريات ورفيق ميكروفلويديك تقنية خلط

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

رد الفعل المحدد رصد الطيف الكتلي لالمطلق البروتين الكمي

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA Electroporation لل، عزل والتصوير من Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

DNA ستراند المهجرين غيتس لشبكات التفاعل التنفيذية الكيميائية المشتقة من البلازميد

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