איורים 1, 2, 3, 4, 6, 8 ולוחות 2, 3, 10, 15, 16 הם שונה מאסמכתא 29. עבודה זו נתמכה על ידי הקרן הלאומית למדע (NSF-להעניק CCF 1,117,143 וNSF-CCF 1,162,141 לGS). י &#39;-JC נתמכה על ידי מלגות ממשלה הטיוואנית. SDR נתמכה על ידי התכנית הלאומית למדע קרן בוגר מחקר המלגה (GRFP).

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The authors declare no competing financial interests.

<html> מאמר זה מתאר שיטה להפקת שערי ndsDNA מפלסמיד דנ&quot;א הטהור ביותר. יתר על כן, פרוטוקול מוצג לאפיון ביצועי שער באמצעות assay קינטיקה הקרינה. נתוני ניסוי מראה כי מערכת הנגזר פלסמיד בביצועיו עמיתו הסינתטי שלה, גם אם המערכת סינתטית מורכבת מחוטים מטוהרים באמצעות ג&#39;ל אלקטרופורזה polyacrylamide (עמוד). סביר להניח, הביצועים המשופרים של שערים הנגזר פלסמיד הוא בעיקר בשל גבוה מאוד טוהר של ה- DNA הביולוגי. DNA סינטטי מכיל מגוון של טעויות, במחיקות מסוימות שתגרומנה oligonucleotides של n-1 אורך, וצד מוצרים אלה בדרך כלל לא הוסרו לחלוטין בעמוד או הליכי טיהור כרומטוגרפיה נוזלית ביצועים גבוהים (HPLC). שיפורים דומים לאלה שדווחו כאן גם נצפו במחקר קודם של מגבר סיכת ראש זרז שהשתמש בדנ&quot;א המופק ממקורות ביולוגיים 21. התחת = &quot;jove_content&quot;&gt; עם זאת, גם השימוש בשערים הנגזר פלסמיד לא יכולים לחסל לחלוטין טעויות בביצועי השער, שיש לפחות שתי סיבות: ראשונה על מערכת עיכול או חוסר הדיוק לחתוך יכול להוביל לשערים עם יותר מדי ניקס או חתכים בעמדות הלא נכונות. בכל מקרה, שערים נוטים יותר להשתתף בתגובות לא רצויות. בעיות מסוג זה עשויים להיות הקלה על ידי אופטימיזציה של כמות האנזים המשמשת (ראה איור 4). שנית, בניסויים אלה, רוב התשומות וגדילי דנ&quot;א סינטטי עזר היו וכך הכילו מחיקות ומוטציות. באופן עקרוני, יכולים להיות גם השיגו את כל הקלט חד-גדילים וגדילי עזר מ- DNA phagemid דרך עיכול אנזים nicking של הגנום הנגיפי M13 קידוד מראש 26. אולי ביצועי המעגל ניתן לשפר עוד יותר באמצעות ssDNA נגזר מהגנום של החיידקים. בעוד שהשימוש בשערים הנגזר פלסמיד נמצא כדי לשפר את ביצועי מעגל, ניתוחשל הפעמים העלות ועיבוד גילה כי בעוד שהייצור של שערים הנגזר פלסמיד הוא מעט זול יותר (לוח 15), זה לוקח 2-3 פעמים יותר זמן עיבוד בהשוואה להרכבה וטיהור של שערים מoligos המסונתזת מסחרי (לוח 16). העלויות העיקריות של שערים הנגזר פלסמיד הן סינתזת גן והשימוש באנזימי הגבלה. עבור 300 pmole של שערים (15 תגובות מספיק ליום 30 בננומטר), העלות המשוערת להצטרפות שערים היא כ 170 $ ו 200 $ עבור מזלג שערים, להיות הבדל העלות בשל השימוש באנזימים שסחבו שונים. לעומת זאת, הסינתזה הכימית של הגדילים לאותו שער העלויות כ 260 $ כוללים דמי טיהור עמוד. עלות הזמן העיקרית לשערים הנגזר פלסמיד היא בתהליך השכפול, ש, בדיוק כמו סינתזת DNA, יכול להיות במיקור חוץ לחברת סינתזת גן. עם זאת, יש לי שערים לאחר הרכבה, הנגזר פלסמיד היתרון שיכולים בקלות להיות משוכפלים פלסמידים המארחניתן לאחסן nd בצורה של מניות גליצרול בקטריאלי. זה מאפשר לעשות שימוש חוזר בשערים פעמים רבות. במבט קדימה, ביצועים המשופרים של שערים הנגזר פלסמיד יכולים לאפשר מגוון הרבה יותר גדול של התנהגויות דינמיקה מאשר הודגם בניסוי עד כה עם ה- DNA CRNs. לדוגמא, 47,48 עבודה תיאורטית האחרון הציע שדפוסים מרחביים באמצעות התארגנות עצמית במקרו בקנה המידה יכולים להתממש עם CRNs DNA באמצעות מנגנון דיפוזיה תגובה. השיטה המוצגת כאן מספקת נתיב קיימא לבניית הרכיבים המולקולריים שבבסיס לחומרי DNA דפוסים עצמיים כזה. למרות מאתגר, פיתוח מורפולוגיות מאקרו בקנה מידה בדרך לתכנות יהיו השלכות משמעותיות בתחומים החל מחקר בביו-חומרים לרפואת רגנרטיבית.

יכולת החיזוי של זיווג הבסיס ווטסון-קריק אפשרה DNA ננוטכנולוגיה הדינמי לצוץ כדרך הניתנת לתכנות לעיצוב התקנים מולקולריים עם תכונות דינמיות 1,2. בפרט, עקירת גדיל DNA - תגובת הכלאת תכנות, תחרותית - הוכיחה את עצמו מנגנון רב עוצמה להנדסת מערכות ה- DNA דינמיות. בתגובת עקירת גדיל DNA, oligonucleotide הנכנס דוחק גדיל קשור בעבר &quot;פלט&quot; משותף מחייב משלים. ניתן לקשר תגובות כגון מרובות יחד לתוך מפלי תגובה רב-שלבים עם רמה גבוהה של שליטה על הסדר והעיתוי של תגובה בודדת שלבים 3. מפלי עקירת גדיל DNA נעשו שימוש כדי ליצור מעגלים דיגיטליים ואנלוגיים מולקולריים 4-7, ננו החלפת 8-10, מנועים אוטונומיים מולקולריים 11-15, ומגברי קטליטי שאינם קוולנטיים 13,16-21. יתר על כן, Dניתן לדמות מכשירי NA באמצעות תגובות עקירת גדיל ומיועדים ליישומים מגוונים באמצעות תוכנת עיצוב בעזרת מחשב 22-24. נכון לעכשיו, ה- DNA מסונתז כימי משמש כחומר העיקרי לננוטכנולוגיה DNA. עם זאת, טעויות בתהליך הסינתזה של DNA, וכתוצאה מכך oligonucleotides מושלם, הם האמינו להגביל את הביצועים של מכשירי ה- DNA דינמיים על ידי גרימת תופעות תגובות שגויות. לדוגמא, תגובות &quot;דליפה&quot; יכולות לגרום לשחרורו של oligonucleotide תפוקה גם בהיעדרו של הדק תגובה. תופעות כאלה הן ברורות ביותר במפלי תגובה עצמן שבו אפילו כמות מזערית של דליפה הראשונית סופו של דבר לגרום להפעלה המלאה של 19,20 המפל. לעומת זאת, תגובות לעתים קרובות אינן מצליחות להגיע לרמה הצפויה של הפעלה כי חלק מרכיבים לא לעורר אפילו בנוכחות של הקלט המיועד 7,25. מבוסס DNA כדי להפוך את הביצועים שלננו-התקנים דומים לעמיתיהם המבוססים על החלבונים הביולוגיים שלהם, מצבי שגיאה כזו צריך להיות מופחת באופן דרמטי. פלסמידים חיידקים או DNA הביולוגי אחר יכולים לשמש כמקור זול יחסית של ה- DNA הטהור ביותר עבור יישומי ננוטכנולוגיה. כמויות גדולות של DNA יכולות להיות שנוצרו על ידי שכפול בחיידקים ויכולות ההגהה הפנימיות של מערכות חיות להבטיח את הטוהר של ה- DNA וכתוצאה מכך. למעשה, כמה מאמרים האחרונים הכירו בשירות הפוטנציאלי של ה- DNA הביולוגי ליישומי ננוטכנולוגיה 21,26-28. עם זאת, הטבע-גדילים כפולים המלאה של פלסמיד דנ&quot;א עד כה אסור שימוש בו כחומר לייצור מכשירי ה- DNA דינמיים, אשר בדרך כלל מורכב מהמספר הרב של oligonucleotides ומכילים שני תחומים פעמים התקועות וחד-גדילים. במאמר האחרון 29 סוגיה זו הייתה ממוענת ואדריכלות שער DNA חדשה שמורכבת בעיקר מגדילי דנ&quot;א כפול חתוכות (ndsDNA) היה להציגד. חשוב לציין, יכולות להיות מתוכננות מערכות שערי ndsDNA שמבינות את הדינמיקה שצוינה על ידי כל רשת פורמאלית תגובה הכימית (CRN) 29. יכולים אפוא לשמש שערי ndsDNA, בעיקרון, כדי ליצור מערכות דינמיות שמפגינות תנודות ותוהו ובוהו, bistability וזיכרון, לוגיקה בוליאנית או התנהגויות אלגוריתמיות 30-38. לדוגמא, אסמכתא. 29 הפגינו CRN שלוש-תגובה שסיפק יישום מולקולרי של פרוטוקול &quot;קונסנסוס&quot;, סוג של אלגוריתם מחשוב מבוזר 29,39,40. עבודה זו הוצגה לראשונה שימוש חדשני לפורמליזם CRN כ&quot; שפת תכנות &quot;למהירות סינתזת מערכות מולקולריות פונקציונליות (איור 1 א). כאן, פרוטוקול מפורט לנובעים שערי ndsDNA מפלסמיד דנ&quot;א מסופק. הראשון הוא סקירה של תהליך עיצוב הרצף. לאחר מכן, בעקבות הסבר של oligonucleotides איך סינטטי המכילרצפי השער הם משובטים לתוך פלסמידים והרצף אומת ומוגבר באמצעות תרבות חיידקים. לאחר מכן, הוא הראה כיצד ניתן לגזור ndsDNA שערים מפלסמידים על ידי עיבוד האנזימטית (ראה איור 2). לבסוף, שיטה לבדיקת שער התנהגות באמצעות מבחני קינטיקה הקרינה מתוארת. מנגנון תגובה כדוגמא, הפרוטוקול מתמקד בתגובה כימית + B-&gt; B + C קטליטי. מיני A, B, ו- C (&quot;אותות&quot;, איור 1) מתאים לכל מולקולת DNA חד-גדילים שונה. הרצפים של מולקולות אלה הם עצמאיים לחלוטין והגדילים לא מגיבים אחד עם השני באופן ישיר. יש הרצפים של כל האותות שני תחומים שונים פונקציונליים, כלומר, subsequences שפועל יחד בתגובות עקירת גדיל: 1) תחום דריסת רגל קצר (תוויות ת&quot;א, שחפת, TC) המשמש לייזום של r עקירת הגדילeaction, ו -2) תחום ארוך (תוויות, B, C) הקובע את זהות האות. אינטראקציות בין גדילי אות מתווכות על ידי מתחמי חתוכות פעמיים גדילי דנ&quot;א (ndsDNA) שער (נקראים הצטרפו AB ומזלג לפני הספירה) ומינים חד-גדילי עזר (&lt;tr r&gt;, &lt;b tr&gt;, &lt;ג TB&gt; ו- &lt;i TC &gt;). התגובה הרשמית + B-&gt; B + C מתבצע באמצעות סדרה של צעדי תגובת עקירת גדיל, שבו כל צעד תגובה חושף דריסת רגל לתגובה הבאה (איור 1). בדוגמא זו, אותות ו- B הם בתחילה בחינם בפתרון תוך C אות קשור לשער המזלג. בסוף התגובה B ו- C הם בפתרון. באופן כללי יותר, אותות שחייבים שער אינם פעילים ואילו אותות שהם חופשיים בפתרון הם פעילים, כלומר, הם יכולים להשתתף בתגובת עקירת גדיל כקלט. במהלך זמן התגובה ואחריו באמצעות אסטרטגית כתב ניאון (איור 1 ג). בעבודה קודמת 29, זה היה הראה כי מנגנון תגובה זו מבין stoichiometry הנכון אלא גם קינטיקה של תגובת היעד לא רק.

<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.

גדיל DNA עקירת עיר רשתות תגובת השרשת כימית המופק מפלסמיד

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 ...

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14.1K 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.

ניתוח תיקון DNA פעמיים גדיל Break (DSB) בתאי יונקים

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.

איך להקליט התרבות לעידוד רשתות נוירונים על מיקרו אלקטרודה מערכים (MEAs)

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.

האדם Bioengineering רשתות כלי הדם של עכברים Immunodeficient

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.

איטרטיבי אופטימיזציה של דופלקסים DNA להתגבשות של מכלולי SeqA-DNA

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.

זיהוי של חיידקים פעילים מטבולית במעיים של Generalist<em&gt; Spodoptera littoralis</em&gt; באמצעות איזוטופ יציב DNA גשוש שימוש<sup&gt; 13</sup&gt; C-גלוקוז

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.

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

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

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.

הדמיה בזמן אמת של יחיד תכנון הנדסי RNA תמלילים בתאים חיים באמצעות משואות bimolecular רציומטרי

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.

הפגנות חומרת הסביבה מופחתת כובד של טכנולוגיה cytometer זרימה וCompanion Microfluidic אב טיפוס מוקטנת ערבוב

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.

תגובה נבחרת ניטור ספקטרומטריית מסה לכימות חלבון מוחלט

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

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

In vivo אלקליין השביט Assay ואנזים-modified אלקליין השביט Assay עבור מדידת Breaks Strand דנ&quot;א נזק חמצוני דנ&quot;א עכברוש הכבד

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...