يود الكاتب أن أشكر كل من بلدي الجامعية المستقلة طلاب البحوث وكل من بلدي الميكروبية الفسيولوجيا طلاب الدراسات العليا الذي اختبر أفكاري ينقول الطفرات خلال فصول الربيع 2010-2014. وقد تم تمويل هذا العمل من قبل قسم العلوم البيولوجية في جامعة ولاية يانجز.

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الكاتب ليس لديه ما يكشف.

الطفرات ينقول باستخدام transposome هي أداة فعالة لتوليد auxotrophs في الأمعائية س. YSU وأنواع أخرى من غرام سلبي وغرام البكتيريا إيجابية 3،4. وقد بدأت هذه العملية عن طريق إدخال transposome -derived تينيسي 5 إلى المضيف بواسطة الصعق الكهربائي. لتحديد المستعمرات مع تدرج، وكان ا...

أنموذجية التغذي البكتيريا تنمو في M-9 الأملاح التي تحتوي على الحد الأدنى من متوسط ​​الجلوكوز (M-9 المتوسطة)، وتحويل الجلوكوز من خلال مسارات الأيض الكربون المركزية لتوليد السلائف، مثل الأحماض الأمينية، والأحماض النووية والفيتامينات، ل1 الحيوي. M-9 المتوسطة يحتوي على كلوريد الأمونيوم كمصدر النيتروجين والفوسفات والبوتاسيوم والصوديوم كمنطقة عازلة ومصدر الفوسفور، كبريتات المغنيسيوم كمصدر الكبريت والجلوكوز كمصدر للكربون والطاقة. لوريا، Bertani (LB) متوسطة غنية في الأحماض الأمينية من تريبتون والفيتامينات وعوامل النمو من خلاصة الخميرة. وهو يدعم نمو auxotrophs التي لا يمكن توليف الأحماض الأمينية والفيتامينات وعوامل النمو الأخرى المطلوبة للنمو على M-9 متوسطة. وبالتالي، سوف prototrophs تنمو في LB M-9 والمتوسطة، في حين auxotrophs سوف تنمو في LB المتوسطة ولكن ليس في M-9 متوسطة. من خلال تقديم الطفرات في عدد السكان بدئية الاغتذاء من البكتيريا وتحديد الجينات الطافرة التي تسبب الظواهر بعامل نمائي، هو بوssible للحصول على فهم أفضل لعملية الأيض في سلالة بكتيرية. ويمكن استخدام ينقول الطفرات لتحديد العديد من الجينات المطلوبة للنمو على مستوى السكر في M-9 متوسطة. الترانسبوزونات إدراج نفسها عشوائيا في جينوم المضيف 2. قبل اكتشاف transformants ينقول على لوحات أجار LB نسخة والطلاء لهم على لوحات M-9 المتوسطة أجار، فمن الممكن للكشف عن auxotrophs. يتم تحديد جينات توقف من خلال إنقاذ الجينات. تستخدم هذه الدراسة متاحة تجاريا تينيسي 5 -derived transposome التي يتم شحنها كحل للشريحة الحمض النووي ينقول الخطي مختلطة مع البروتين ترانسبوزاز. قطاع DNA تفتقر إلى الجين ترانسبوزاز ولكن يحتوي على الجينات المقاومة الكانامايسين، وهو R6K تكرار γ المنشأ واثنين من الزخارف الفسيفسائية التي هي تسلسل الحمض النووي لترانسبوزاز ملزم في نهاية كل قطعة 3،4. منذ يتم إضافة بروتين ترانسبوزاز مباشر على الحمض النووي، والجزء الحمض النووي وحدهيعرف بأنه ينقول، ويعرف مجمع بروتين DNA / ترانسبوزاز باسم transposome. وحولت transposome بواسطة الصعق الكهربائي 5 إلى المضيف حساسة الكانامايسين (الشكل 1A). المستعمرات التي تنمو على لوحات أجار LB تحتوي على كانامايسين (LB-اساسه) لديها إدراج ينقول (الشكل 1B)، ونسخة مطلية transformants التي لا تنمو على M-9 لوحات المتوسطة أجار التي تحتوي على كانامايسين (M-9-اساسه) هي auxotrophs (Figire 1C). الحمض النووي الجيني من المسخ هو النقى ويهضم جزئيا مع 4-قاعدة تقييد القطع نوكلياز داخلية، الاتحاد البلغاري CI (الشكل 1D). يتم تحويل الحمض النووي و ligated الى سلالة من القولونية (إي كولاي) الذي يحتوي على الجينات البير (الشكل 1E). يسمح هذا الجين البلازميد جديد يحتوي على ينقول والمنطقة المحيطة الكروموسومات المضيفة لتكرار في E. القولونية 6. ويعمل هذا الجين المقاومة الكانامايسين مثلعلامة جدير بالانتخاب لالبلازميد الجديد. أخيرا، تسلسل باستخدام بادئات مكملة لبعضها نهاية ينقول والأساسية المحلية محاذاة أداة البحث (انفجار) 7،8 تحليل تسلسل الناتجة تستخدم لتحديد هوية الجينات توقف. وتنص هذه الاستراتيجية transposome الطفرات ثلاث مزايا 3. أولا، لأن البروتين ترانسبوزاز لا بد مباشرة إلى ينقول، الإدراج لا يعتمد على التعبير عن الجينات ترانسبوزاز داخل المضيف. مرة واحدة يدمج ينقول نفسه في جينوم المضيف، مع تدهور ترانسبوزاز، ومنع حركة إضافية لينقول. ومع ذلك، حركة إضافية لا يمكن منعها إذا كان المضيف يمتلك الذاتية عنصرا transpositional تينيسي 5. ثانيا، إدخال transposome بواسطة الصعق الكهربائي يجعل من الممكن استخدامها في مجموعة واسعة من المضيفين. كما أنه يزيل الحاجة إلى إدخال ينقول طريق الاقتران البكتيري أو السادسإصابة راؤول. تتطلب كلتا العمليتين قابلية المضيف. ثالثا، إدراج الجينات المقاومة الكانامايسين وR6K تكرار γ الأصل في transposome يجعل من الاسهل لتحديد الجين توقف. المناطق ينقول انقطاع ويمكن تخزين والتسلسل كما البلازميدات، مما يلغي الحاجة إلى استخدام سلسلة رد فعل البلمرة العكسية (PCR) تقنية لتحديد الجينات. بروتوكول المعروضة في هذا الفيديو يصف كل خطوة لينقول الطفرات من الأمعائية س. YSU 9 باستخدام transposome من عرضه في الخلايا البكتيرية إلى تحديد الجين المفترض أنها توقفت. بالإضافة إلى البروتوكولات نشرت سابقا 3،4،10، يتم عرض طرق مفصلة لاستخدام الطلاء طبق الأصل للكشف عن auxotrophs. ويمكن استخدام هذه التقنية الطفرات عن التحقيق في الظواهر الأخرى، مثل المضادات الحيوية والمعادن المقاومة، في أنواع مختلفة من البكتيريا، لIDENTifying عدد ضئيل من الجينات المطلوبة للنمو في ظل ظروف ثقافة المحددة في دراسات علم الأحياء الاصطناعية، أو لتعليم عنصرا مختبر لعلم الوراثة أو علم وظائف الأعضاء بالطبع الميكروبي.

<table border="0" cellpadding="0" cellspacing="0" width="707">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:227px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EZ-Tn5 R6K&#947;ori/KAN-2 Tnp Transposome Kit</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">TSM08KR</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EC100D pir+ Electrocompetent E. coli</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">ECP09500</td>
			<td style="width:227px;">Capable of replicating plasmids with an R6K&#947; replication origin at a low copy number</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EC100D pir-116 Electrocompetent E. coli</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">EC6P095H</td>
			<td style="width:227px;">Capable of replicating plasmids with an R6K&#947; replication origin at a high copy number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5X M-9 Salts</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">DF048517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lennox LB Broth</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">BP1427-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agar</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J637-1KG</td>
			<td style="width:227px;">Solid media contained 1.6% (w/v) agar</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kanamycin Sulfate&#160;</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">0408-25G</td>
			<td style="width:227px;">When required, media contained 50 &#956;g/ml kanamycin sulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-Glucose Monohydrate</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">0643-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J850-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptone</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J859-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4504-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">X190-1KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgCl2</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:119px;">BP214-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO2</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:119px;">BP213-1</td>
			<td></td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:216px;">Super Optimal Broth with Catabolite Repression (SOC) medium</td>
			<td style="width:145px;"></td>
			<td style="width:119px;"></td>
			<td style="width:227px;">0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4 and 20 mM Glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BfuC I</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0636S</td>
			<td>Partial digestion of genomic DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xho I</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0146S</td>
			<td>Plasmid digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA Ligase</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nuclease Free Water</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">E476-1L</td>
			<td style="width:227px;">For dissolving precipitated DNA and restriction endonuclease reactions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GenomeLab DTCS - Quick Start Kit</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:119px;">608120</td>
			<td>DNA sequencing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:145px;">Promega</td>
			<td style="width:119px;">A1120A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wizard Plus SV Minipreps DNA Purification System</td>
			<td style="width:145px;">Promega</td>
			<td style="width:119px;">A1460</td>
			<td>Plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica Plating Block</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">09-718-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen Squares</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">09-718-2</td>
			<td>Replica plating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PetriStickers</td>
			<td style="width:145px;">Diversified Biotech</td>
			<td style="width:119px;">PSTK-1100</td>
			<td>Grid for replica plating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri Dishes</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser II</td>
			<td style="width:145px;">BioRad</td>
			<td style="width:119px;"></td>
			<td>Electroporation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electroporation Cuvettes &#8211; 2 mm</td>
			<td style="width:145px;">BioExpress</td>
			<td style="width:119px;">E-5010-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CentriVap DNA Vacuum Concentrator</td>
			<td style="width:145px;">Labconco</td>
			<td style="width:119px;">7970010</td>
			<td>Drying DNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CEQ 2000XL DNA Analysis System</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td>DNA sequencing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vector NTI Advance 11.5.0</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">12605099</td>
			<td>DNA sequence analysis</td>
		</tr>
	</tbody>
</table>

generation of enterobacter sp. ysu auxotrophs using transposon mutagenesis

أنموذجية التغذي البكتيريا تنمو على M-9 الأملاح الحد الأدنى تستكمل المتوسطة مع الجلوكوز (M-9 المتوسطة)، والذي يستخدم كمصدر للكربون والطاقة. يمكن أن تتولد Auxotrophs باستخدام transposome. المتاحة تجاريا، TN 5 transposome -derived المستخدمة في هذا البروتوكول يتكون من شريحة خطي من الحمض النووي الذي يحتوي على R6K أصل النسخ γ، جين المقاومة الكانامايسين واثنين تسلسل الفسيفساء تنتهي، والتي تكون بمثابة ترانسبوزاز ملزمة المواقع. وtransposome، على النحو المنصوص عليه مجمع بروتين DNA / ترانسبوزاز، هو عرض بواسطة الصعق الكهربائي في سلالة بدئية الاغتذاء، الأمعائية س. YSU، ويشتمل على نفسها عشوائيا في جينوم هذا المضيف. Transformants هي نسخة مطلية على لوريا، Bertani أجار لوحات تحتوي على كانامايسين، (LB-اساسه) وعلى لوحات أجار M-9 متوسطة تحتوي على كانامايسين (M-9-اساسه). تعتبر transformants التي تنمو على لوحات LB-اساسه ولكن ليس على لوحات M-9-أن يكون اساسه auxotrophs. الجينومية النقىيتم هضم الحمض النووي من عوني التغذي جزئيا، و ligated وتحولت الى البير + الإشريكية القولونية (إي كولاي) سلالة. تكرار أصل R6Kγ يسمح البلازميد إلى تكرار في البير + E. سلالات القولونية، والمقاومة علامة كانامايسين تسمح لاختيار البلازميد. كل المحولة يمتلك البلازميد جديد يحتوي على ينقول يحيط بها المنطقة الكروموسومات انقطاع. سانجر تسلسل والأساسية المحلية محاذاة أداة البحث (انفجار) تشير إلى الهوية المفترضة من الجين توقف. هناك ثلاث مزايا لاستخدام هذه الاستراتيجية الطفرات transposome. أولا، أنها لا تعتمد على التعبير عن الجينات ترانسبوزاز من قبل المضيف. الثاني، هو عرض transposome إلى المضيف الهدف بواسطة الصعق الكهربائي، وليس عن طريق الاقتران أو عن طريق التنبيغ وبالتالي أكثر كفاءة. ثالثا: تكرار أصل R6Kγ يجعل من السهل تحديد تحور زالشم الذي تعافى جزئيا في البلازميد المؤتلف. هذه التقنية يمكن استخدامها للتحقيق في الجينات المسؤولة عن الخصائص الأخرى للالأمعائية س. YSU أو من مجموعة متنوعة من السلالات البكتيرية.

تحويل الأمعائية س. YSU بواسطة الصعق الكهربائي مع transposome بدأت عشوائي الجينوم الإدراج في جينوم المضيف (الشكل 1A، B). وElectroporation للنجاح أثمرت عدة آلاف من transformants التي نمت على لوحات LB-اساسه. للحصول 300-400، المستعمرات في لوحة متباعدة بشكل جيد، تم تحسين كمية انتشار خل...

Watch this Scientific Journal Video about Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis at JoVE.com

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

الأمعائية س. YSU تنمو في الجلوكوز الحد الأدنى من الأملاح المتوسطة. يتم إنشاء Auxotrophs عن طريق تحويل ذلك مع transposome الذي إدراج نفسها عشوائيا في جينوم المضيف. تم العثور على المسوخ بواسطة الطلاء المتماثلة من المتوسطة معقد إلى الحد الأدنى من المتوسط. يتم تحديد جينات الانقاذ توقفت بسبب الجينات وتسلسلها.

جيل من<em&gt; الأمعائية</em&gt; س. YSU Auxotrophs عن طريق الطفرات ينقول

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

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Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

13.8K Views.  Youngstown State University.  الأمعائية س. YSU تنمو في الجلوكوز الحد الأدنى من الأملاح المتوسطة. يتم إنشاء Auxotrophs عن طريق تحويل ذلك مع transposome الذي إدراج نفسها عشوائيا في جينوم المضيف. تم العثور على المسوخ بواسطة الطلاء المتماثلة من المتوسطة معقد إلى الحد الأدنى من الم...

Video: Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

توليد مستقرة ايليجانس جيم المعدلة وراثيا عن طريق Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

الموقع محلية الصنع تطفير موجه من البلازميدات الجامعة

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

جيل من الحمض النووي الريبي / الحمض النووي DNA في الجينوم الهجائن التي تحول باستخدام الحمض النووي الريبي التي تحتوي على [أليغنوكليوتيد]

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

مقدمة لمختبر دودة : من الديدان إلى التثقيف الطفرات

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

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 ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

وحيد الطفرات سيت بيسلفيت

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

سريعة وفعالة الزرد التنميط الجيني باستخدام PCR عالية الدقة مع تحليل نذوب

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

كفاءة الجذع الجيل خلية من الدم عن طريق Episomes وHDAC المانعون

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

بروتوكول لالتقييم الوظيفي من كامل البروتين التشبع الطفرات المكتبات الاستفادة من الإنتاجية العالية التسلسل

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...