חומר זה מבוסס על עבודה נתמכת על-ידי Office של המחקר הימי פרס מספר N00014-13-1-0074 (V.N.), תוכנית המדע הגבול האנושי להעניק מספר RGP0037/2015 (V.N.), הקרן הדו-לאומית למדע להעניק 2014400 (V.N.).

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+prototyping+genetic+networks+with+cell-free+transcription-translation+reactions.">Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.</a> Methods. , (2015).</li><li>Shin, J., Jardine, P., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Replication,+Synthesis,+and+Assembly+of+the+Bacteriophage+T7+in+a+Single+Cell-Free+Reaction.">Genome Replication, Synthesis, and Assembly of the Bacteriophage T7 in a Single Cell-Free Reaction.</a> ACS Synthetic Biology. 1 (9), 408-413 (2012).</li><li>Garamella, J., Marshall, R., Rustad, M., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+All+E.+coli+TX-TL+Toolbox+2.0:+A+Platform+for+Cell-Free+Synthetic+Biology.">The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.</a> ACS Synth Biol. , (2016).</li><li>Karzbrun, E., Shin, J., Bar-Ziv, R. 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המחברים מצהירים את interest(s) הפיננסיים מתחרים הבאים: מעבדה Noireaux מקבל וקרנות מחקר MYcroarray, מפיץ של ערכת ביטוי חלבון נטול תא MYtxtl.

בעקבות הטכניקה חן, et al. 14 עבור SPMC, שלב קריטי נגיש בקביעת תנאי נאותה superinfection. הפרמטר השולט ביותר היכולת של זן מארח לעמוד superinfection הוא לעתים קרובות הריכוז ההתחלתי של phage המזהם. התאים מארח חייב להיות בשלב הצמיחה לוגריתמי לפני זיהום ראשוני עם כמות קטנה מאוד של phage. בסופו של דבר, pha...

בעשור האחרון, הטכנולוגיה ביטוי נטולת תא מהונדס כתובת יישומים חדשניים בביולוגיה הקשור סינטתיים, כמותיים אזורי מחקר רב-תחומיים מתהווה. השתמשו במקור לבטא חלבונים עצמאית של אורגניזם חי, מערכות TXTL נטול תאים חדשים פותחו שני מדעי בסיסי ויישומי1,2, מרחיבה משמעותית את ההיקף בטכנולוגיה זו. הדור החדש של פלטפורמות TXTL תוכנן להיות ידידותי למשתמש, יעיל יותר (בדרך להשגת 2 מ"ג/מ"ל של סינתזת חלבונים אצווה מצב3), יותר ברמה של שעתוק4, ועם זאת מודולרי על מנת לשלב בקלות הרומן טבעי או פונקציות סינתטי להרחיב את היכולות של מערכות ביולוגיות קיימות5,6. בפרט, ללא תא TXTL מערכות הפכו להיות שימושי עבור שטנץ מהירה של תוכניות גנטי, כגון גורמים רגולטוריים או המעגלים גנטי קטן7,8,9, על-ידי הפחתת העיצוב-לבנות-המבחן מחזור מספר ימים. למרבה הפלא, המערכות TXTL חדש גם הם מסוגלים עיבוד תוכניות דנ א גדולים כגון הסינתזה מלאה של coliphages10,11, הוכחת חזקה מספיק הופעות כדי לתמוך את בנייתו מחדש של פעיל גנומית ה-DNA מקודד ישויות החיים.
TXTL מערכות להציג יתרונות טכניים רבים לעומת המסורתי במבחנה בונה הביוכימי מבחני. ללא תא TXTL מקשר את התהליך של ביטוי גנים המוצר הסופי בסביבה מופחתת ופתוח, בניגוד הציטופלסמה מורכבת של תא חי. TXTL משתמש דנ א כדי לשחזר מערכות ביוכימיות במבחנה, באמצעות טכניקות מודרניות של מכלול ה-DNA, וזה, במחיר נוח, מהיר בנוסף לדרישה לא בררן חלבון טיהור צעדים. תא ללא ביטוי מספקת גישה ישירה אל רוב הרכיבים השונים תגובות ביוכימיות, ובכך מאפשר ניתוח עמוק יותר אינטראקציות מולקולרית12. בתגובה TXTL, אחד באפשרותך לשנות את ההגדרות הביוכימי, ביופיזיקלי כרצונו, שבו כמעט בלתי אפשרי בתא החי. לאור יתרונות ושיפורים אחרונים אלה, הטכנולוגיה TXTL הופכת לפופולרית יותר ויותר כמו פלטפורמה חלופי עבור ביולוגיה סינתטית ולא כמותית. בעוד קהילת המחקר באמצעות TXTL מתפתחת במהירות, TXTL הופך להיות טכנולוגיה סטנדרטית בביו-הנדסה, זה חיוני כדי להבין כיצד משתמשים כזה פלטפורמות כדי לפתח נאותה שהיו קשורים ההוצאה להורג של תגובות TXTL ולחוות פרשנות של התוצאות.
במאמר זה, אנו מסבירים כיצד להשתמש במערכת TXTL כל של החיידק לסנתז, בתגובות סיר אחד, bacteriophages מ שלהם הגנום11, כגון MS2 (RNA, 3.4 kb), ΦΧ174 (ssDNA, 5.4 kb), ו- T7 (dsDNA, 40 kb). אנו מראים איך כמות phages מסונתז שינויים ביחס כמה הגדרות הביוכימי של התגובות (ריכוזים מגנזיום ואשלגן). הצפיפות המולקולרית, מדומה דרך ריכוזים טווח של פג 8000, יש השפעה דרמטית על סינתזה phage במספר סדרי גודל. מימוש מערכות הביוכימי גדולים כאלה בתגובות מבחנה אחת אשר מסכם את הדברים במקביל את התהליכים של תמלול, תרגום, הרכבה עצמית, מעניין למיעון שאלות בסיסיות הקשורות לביולוגיה וביופיזיקה 10 (הכונה הרכבה עצמית), כמו גם עבור פיתוח יישומים, כגון שינוי פונקציות phage לבנות nanostructures חדש13. בנוסף מעשית על TXTL, אנו מספקים שיטות phage הגברה, חילוץ הגנום, טיהור של כימות phage מאת וזמינותו פלאק. השיטות שהוצגו בכתב היד מתאימים לחוקרים להשתמש במערכות ללא תא תמצית מבוסס e. coli , מעוניינים bacteriophages.
הפרוטוקולים הציג בעבודה זו ניתן לסכם כדלקמן: 1) Phage הגברה (יום 1: להכין חיסון תאי, יום 2: יחיד פלאק, מרובים phage צמיחה, ואת לריכוז יום 3: טיהור של phage), 2) מיצוי DNA הגנום גדילי כפול (פנול/כלורופורם מיצוי), ו-3) ללא תא התגובה phage וניסוי כייל נוגדנים (יום 1: צלחת התאים המארחים ולעשות פלטות אגר, יום 2: התגובה ללא תא ומנחה תא תרבות קדם, ו יום 3: מארח תא phage ותרבות כייל נוגדנים).

<table width="899">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:168pt" width="224" />
		<col style="width:219pt" width="292" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:162pt;height:15.75pt" width="216">Ultracentrifugation tubes</td>
			<td class="xl21" style="width:168pt" width="224">Beckman Coulter</td>
			<td class="xl21" style="width:219pt" width="292">344057</td>
			<td class="xl20" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Conical tubes</td>
			<td class="xl22" style="width:168pt" width="224">Falcon</td>
			<td class="xl21">352070</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Gradient maker</td>
			<td class="xl21">BioComp Gradient Master</td>
			<td class="xl22" style="width:219pt" width="292">see Anal Biochem. 1985 Jul;148(1):254-9.</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Syringe and Blunt Cannula</td>
			<td class="xl21">Monoject</td>
			<td class="xl21">8881513918 and 888202017</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Wide-bore pipette tips</td>
			<td class="xl21">Fischerbrand</td>
			<td class="xl21">02-707-134</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Plaque counter</td>
			<td class="xl21">New Brunswik Scientific</td>
			<td class="xl21">Colony Counter Model C-110</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Culture tubes</td>
			<td class="xl22" style="width:168pt" width="224">Fischerbrand</td>
			<td class="xl21">14-961-33</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Cell-free system</td>
			<td class="xl22" style="width:168pt" width="224">Mycroarray Inc</td>
			<td class="xl22" style="width:219pt" width="292">Mytxtl</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">BioComp Gradient Master</td>
			<td class="xl17" style="width:168pt" width="224">BioComp Instruments</td>
			<td class="xl17" style="width:219pt" width="292">Model 105ME</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">LB agar plate recipe</td>
			<td class="xl20"></td>
			<td class="xl20"></td>
			<td class="xl24" style="width:512pt" width="683">25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td>
		</tr>
	</tbody>
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synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

דור חדש של מערכות ללא תא תמלול-תרגום (TXTL), מתוכנן יש רב-תכליתיות גדולה יותר, מודולריות, לספק יכולות הרומן לבצע מדעי בסיסי ויישומי בתגובות מבחנה. בעשור האחרון, ללא תא TXTL הפך טכניקה חזקה עבור מגוון רחב של מחקר רב תחומי חדשניים בתחומים הקשורים כמותיים וסינתטיים ביולוגיה. פלטפורמות חדשות TXTL שימושיים בעיקר לבנות ולחקור מערכות הביוכימי דרך הביצוע של מעגלים ג'ין סינטטי או טבעי. במבחנה TXTL הוכיחה נוח במהירות רכיבי רגולטוריות אב-טיפוס, רשתות ביולוגיות כמו גם באשר לסכם מולקולרית הרכבה עצמית מנגנונים נמצאו במערכות החיים. במאמר זה, אנו מתארים איך זיהומיות bacteriophages, כגון MS2 (RNA), ΦΧ174 (ssDNA), ו- T7 (dsDNA), הם לגמרי מסונתז מן הגנום שלהם בתגובות סיר אחד באמצעות של Escherichia coliכל, ללא תאים של מערכת TXTL. סינתזה של coliphages שלושה זה לכמת באמצעות וזמינותו פלאק. אנו מראים איך התשואה של phage מסונתז תלוי בהגדרות הביוכימי של התגובות. הצפיפות המולקולרית, מדומה דרך ריכוז מבוקר של פג 8000, משפיעה על כמות phages מסונתז על ידי הוראותיו של מגניטודות. אנו נסביר גם כיצד להגביר את phages ואיך לטהר את הגנום שלהם. קבוצת פרוטוקולים ותוצאות הציג בעבודה זו צריך להיות עניין רב תחומיים החוקרים המעורבים ב ביולוגיה סינתטית ללא תא וב בביו-הנדסה.

אנו מראים תוצאות נציג ארבע. איור 1, אנו מציגים קבוצה של פקדים שלילי כדי להבטיח כי מערכת TXTL ללא תאים, המניות DNA phage לא נגועים עם חיים התאים e. coli . אנו מאמתים כי מערכת TXTL נטולת תא הינה ללא פגע e. coli תא זיהום על ידי ציפוי שני התגובה שאינו מתפשט, מודגרות פתר...

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

סינתזה של Bacteriophages זיהומיות e. coli -תא ללא ביטוי מערכת מבוססת

דור חדש של פלטפורמות תרגום תמלול חופשי תא תוכנן לבנות מערכות ביוכימיות במבחנה באמצעות הביצוע של ג'ין מעגלים. במאמר זה, אנו מתארים איך הם bacteriophages, כגון MS2, ΦΧ174 ו- T7, מסונתז מן הגנום שלהם באמצעות e. coli כל ללא תא TXTL המערכת.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Research

JoVE Journal

Bioengineering

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

14.5K Views.  University of Minnesota. דור חדש של פלטפורמות תרגום תמלול חופשי תא תוכנן לבנות מערכות ביוכימיות במבחנה באמצעות הביצוע של ג'ין מעגלים. במאמר זה, אנו מתארים איך הם bacteriophages, כגון MS2, ΦΧ174 ו- T7, מסונתז מן הגנום שלהם באמצעות e. coli כל ללא תא TXTL המערכת.

Video: Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

גילוי חיידקים וזיהוי באמצעות חיישנים אלקטרוכימיים

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

סינתזה של חלבון מלאכותי בתיווך intein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

טכניקה כירורגית הרומן כבסיס In Vivo חלקי הכבד הנדסה בעכברוש

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

תא ללא חלבון ביטוי באמצעות החיידק גדל במהירות ויבריו natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

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

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

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

פלטפורמת מיקרופלואידיג רב שכבתית לצורך הובלה של ביטוי גנים ממושך ללא תא

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

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

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

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

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

זיהוי תפוקה גבוהה של עמידות לפסאודומונס syringae pv. עגבניה בעגבניה באמצעות שתיל שיטפון שיטת

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

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

מערכת במבחנה לאמוד את היעילות התרומבוליטית של היסטוטרפסיה וסם ליטיק

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

מערכת ללא תאים טהורים של OnePot

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

אפיון מהיר של חלקים גנטיים עם מערכות נטולות תאים

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.