המחברים מבקשים להודות מלקולם קמפבל וטוד Eckdahl עם קונסורציום הגנום להוראת פעילים (GCAT) והברית החינוך HHMI-מדע - ציידים הפאג קידום ג&#39;נומיקס ומדע אבולוציונית (SEA-פאגים) תוכנית. פרויקט זה מומן על ידי תרומות של המרכז הלאומי לחקר משאבים (P20RR016460) לבין המכון הלאומי למדעי רפואה כלליים (P20GM103429) מן המכון הלאומי לבריאות. מחקר זה מומן בחלקו על ידי הקרן הלאומית למדע תחת IIA-1457888 # מענק. בנוסף מוסדיים (אואצ&#39;יטה המטביל אוניברסיטת) קרנות נמסרו דרך מלגת מחקר קיץ JD פטרסון.

תכנון 1. Oligonucleotides הזמין עם הקצוות הדביקים המתאימים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה terminators Rho-עצמאית פוטנציאליים באמצעות ניתוח גנטי באמצעות תוכניות שכרגע מחוברים לאתר גלוי. 7 </li><li style=";text-align:right;direction:rtl"> כשעובדים עם DNA גדילי כפול, לקבוע את הכיוון של terminator להיבדק. 7 הפלסמיד-כחול PGR רק מאמתת terminators ligated ב 5 &#39;ל 3&#39; כיוון על החלק העליון (גדיל קדימה). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> המרת terminator גדיל תחתון (הפוך) כדי להשלים התהפך שלה לארגון מחדש של הרצף לבדיקות PGR-הכחולה באמצעות תוכנה ברשת בחינם, למשל, APE. </li></ol></li><li style=";text-align:right;direction:rtl"> לאחר האורינטציה נקבעת כנכון, להוסיף את הקצה הדביק &quot;5&#39;-CGAC-3&quot; &quot;ל -5&quot; סוף הגדיל העליון. מוסיף את הקצה הדביק &quot;5&#39;-CCGC-3&quot; &quot;ל -5&quot; סוף הגדיל התחתון. 8 פעולה זו תבטיח כי כנס ligated יוכנס המתאיםאוריינטציה באמצעות GGA. </li><li style=";text-align:right;direction:rtl"> לאחר רצפים נקבעים, להורות כמו oligonucleotides גדילי יחיד מסחרי. </li></ol> 2. Oligonucleotides חישול (חל רק על מיובשים בהקפאה DNA) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Re- להשעות oligonucleotides בודדים במים חינם nuclease לריכוז של 100 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> הפוך חיץ חישול 10x: 1 M NaCl ו 100 mM Tris-HCl pH 7.4. חיץ חישול יכול להיות מאוחסן על 4 מעלות צלזיוס במשך כמה שבועות. לאחר חיץ חישול הוא עשה oligonucleotides מחדש תלוי, להתחיל את החישול 8. </li><li style=";text-align:right;direction:rtl"> עבור כל terminator להיבדק, להוסיף 16 μl של מים טהורים כדי צינור 1.5 מ&quot;ל microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 2 μl של חיץ חישול 10x על צינור אחד microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של oligonucleotide הגדיל העליון (100 מיקרומטר) מן terminator הרצוי. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של oligonucleotide הגדיל התחתון (100 מיקרומטר) מן terminator הרצוי. </li><li style=";text-align:right;direction:rtl"> צור ba מים רותחיםה באמצעות מבחנה 1,000 מ&quot;ל המכיל כ 400 מ&quot;ל של מי ברז ומניחים על צלחת חמה. </li><li style=";text-align:right;direction:rtl"> מניחים את הצינורות microcentrifuge משלב 2.6 ב לצוף ומרתיחים במשך 4 דקות. לאחר 4 דקות, לכבות את צלחת חום אבל להשאיר את הצינור לצוף באמבטיה מים O מגניב לאט / N (18 שעות) 8. אחסן את terminators annealed ב -20 ° C. </li></ol> 3. אסיפת שער זהב (Mix הרגיל - עלות יעילה) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך דילול 40 ננומטר של terminators המרותק. להוסיף 124 μl של מים nuclease חופשי 1 μl של תמהיל terminators annealed (100 מיקרומטר). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה דגימות עבור 30 שניות ב XG 10,000 ולאחסן על הקרח. </li></ol></li><li style=";text-align:right;direction:rtl"> לתוך מתאים צינור חדש עבור thermocycler בשימוש, להוסיף 6 μl של מי nuclease חינם. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של מסחרי DNA האנזים הצפת [300 mM Tris-HCl (pH 7.7-7.8), 100 מ&quot;מ MgCl 2, 100 מ&quot;מ DTT, ו -10 מ&quot;מ ATP]. ביצוע כל הצעדים הבאים על קרח. </li><li style=";text-align:right;direction:rtl"> לְהוֹסִיף1 μl של PGR-כחול (35-50 ng / μl) פלסמיד היעד אל הצינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של terminators annealed 40 ננומטר אל הצינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 0.5 endonuclease הגבלת μl המסחרי באיכות הגבוהה BsaI אל צינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 0.5 μl של אנזים דנ&quot;א המסחרי אל צינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה תערובת התגובה ב XG 10,000 עבור 120 שניות. אז במקום צינור ישירות לתוך thermocycler. </li><li style=";text-align:right;direction:rtl"> הפעל את התוכנית thermocycler: 20 מחזורים של 1 דקות ב 37 ° C ואחריו 1 דקות ב 16 מעלות צלזיוס, ואחריו 1 מחזור של 15 דקות ב 37 מעלות צלזיוס. השתמש דגימות מיד או להקפיא ולאחסן ב -20 מעלות צלזיוס. 8 </li></ol> 4. אסיפת שער זהב (מיקס מאסטר מסחרי - זמן יעיל) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להוסיף 124 μl של מים nuclease חופשי 1 μl של 100 מיקרומטר annealed terminators שנוצרה בשלב 2. זה מדלל את ריכוז ה- DNA terminator ל -40 ננומטר.בצע פעולה זו בדרך חדשה, שכותרתו צינור 1.5 מ&quot;ל microcentrifuge. </li><li style=";text-align:right;direction:rtl"> לתוך מתאים צינור חדש עבור thermocycler, מוסיף 14 μl מי nuclease חינם. ודא הצינור החדש מסומן בהתאם. </li><li style=";text-align:right;direction:rtl"> הוסף 2 μl של מסחר מיקס מאסטר הצפת צינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של פלסמיד PGR-כחול (35-50 ng / μl) אל הצינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 2 μl של terminators annealed 40 ננומטר אל הצינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> הוסף 1 μl של מסחר מיקס מאסטר אל צינור microcentrifuge. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה הצינור 40 ננומטר ב XG 10,000 עבור 120 שניות. אז במקום צינור ישירות לתוך thermocycler. </li><li style=";text-align:right;direction:rtl"> הפעל את התוכנית thermocycler: 1 מחזור של 60 דקות ב 37 ° C ואחריו 1 מחזור של 15 דקות ב 55 מעלות צלזיוס. השתמש דגימות מיד או להקפיא ולאחסן ב -20 מעלות צלזיוס. </li></ol> טרנספורמציה 5. בחירת קולוני <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכין המאגר לוריא (LB) אגר צלחות פטרי המכילות 1 מ&quot;מ תרכיזיםיון של אמפיצילין (AMP) וכן ריכוז 10 מ&quot;מ של arabinose (ARB). הכן לפחות 10 מ&quot;ל של 1 M L -arabinose פתרון המניות, כי פתרון המניות זה ישמש צעדים מאוחר יותר. </li><li style=";text-align:right;direction:rtl"> השתמש בכל יעילות גבוהה (אוגוסט 10 - ספטמבר 10 transformants / מיקרוגרם) כימי E. המוסמכת coli (JM109) תאים לטרנספורמציה. 9 לקבלת התוצאות הטובות ביותר, להוסיף 3-5 μl של תגובת הקשירה 50 μl של תאים מוסמכים. בעת שימוש תאים מוסמכים כימיים, לעקוב אחר פרוטוקול השינוי ספציפי לתא בשימוש. </li><li style=";text-align:right;direction:rtl"> מורחים מחצית (25 μl) של תאים טרנספורמציה על מחומם מראש LB (Amp / ARB) צלחות אגר באמצעות &quot;מקל הוקי&quot; סטרילי בצורת L דגירה O / N ב 37 מעלות צלזיוס. בשל קצב הפירוק המהיר של אמפיצילין, לא לדגור על 37 מעלות צלזיוס במשך 24 שעות. </li><li style=";text-align:right;direction:rtl"> לבצע בחירת מושבה המבוססת על צבע על ידי בדיקה ויזואלית. קשירה מוצלחת צריכה להיות לבנה / צהובה אור הנראה לזרוח ירוק undeכחול r (450 ננומטר) או אור UV. קשירה מוצלחת תפיק מושבה כי הוא בצבע כחול תחת אור הנראה לאחר 18 - 20 שעות. </li></ol> אימות 6. וכימות של Terminator <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 5 מ&quot;ל צינורות LB-מרק סטרילית עם אמפיצילין 1 מ&quot;מ ו 10 מ&quot;מ arabinose. מספר הצינורות הדרושים תלוי במספר terminators הנבדקת בתוספת שולטת (תאים עם התנגדות אמפיצילין שאינו לזרוח ותאים המכילים פלסמיד PGR-הכחול נימול). </li><li style=";text-align:right;direction:rtl"> תחת אור הנראה לבחור 2-3 מושבות לבנות / צהובות (שלב 5.4) באמצעות לולאה סטרילי. אפשר דגימות כדי לדגור broths על שייקר ב 37 ° C ו 160 סל&quot;ד במשך 16-18 שעות. אין דגירת דגימות אלה במשך יותר מ -24 שעות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחילופין, השתמש ספקטרופוטומטר לקבוע צפיפות התאים אופטי ב 600 ננומטר (OD 600) כדי להבטיח צמיחת תאים נאותה. באופן כללי, OD של 600 ~ 0.8-1.0 הוא ציין לאחר 16-18 שעות של צמיחה. </li></ol></li><li style=";text-align:right;direction:rtl"> להשתמשצלחת 96-היטב סטרילי עבור השלבים הנותרים. לנהל את הפעולות הבאות בסביבה היגיינית בשלושה עותקים. להוסיף 199 μl של מרק LB + Amp / arabinose סטרילית (1 מ&quot;מ אמפיצילין / 10 מ&quot;מ arabinose) זה טוב. כלול חדר על הצלחת במשך שלוש בקרות נפרדות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש בפקדים שלוש הבאות: באר (1) המכיל LB + Amp / arabinose מרק רק ובאר (2) עבור תאים המוסמכות untransformed (ריק) כדי לשמש ריק. עבור כימות (3) של כוח terminator, התאים המכילים PGR המקורי נימול או PGR-כחול המכיל ligated עם רצף הפסקת עישון צריך להיות כלול (שליטה התייחסות כימות). ראה איור 1 עבור פריסת קורא צלחת. </li></ol></li><li style=";text-align:right;direction:rtl"> Pipet 1 μl של פתרון התא מן O / broths N לתוך בארות בודדות של הצלחת 96-היטב. </li><li style=";text-align:right;direction:rtl"> צלחות חותמות עם כיסוי לנשימה ולנער עבור 18-20 שעות ב 37 מעלות צלזיוס, 160 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> באמצעות קורא צלחת, לקבוע OD 600 על ידי קריאהספיגה ב 600 ננומטר. מדוד GFP קרינה כדלקמן: קרינת RFP מדוד עירור 395 ופליטת 509 כדלקמן: עירור 575 ופליטה 605. לאחר 18-20 שעות, OD 600 צריך להיות כ 0.5. </li><li style=";text-align:right;direction:rtl"> כדי לנרמל עבור וריאציה בצמיחה, השתמש המשוואה: <img alt="משוואה 1" src="/files/ftp_upload/54064/54064eq1.jpg" /> הן GFP ו RFP. </li><li style=";text-align:right;direction:rtl"> לאחר נורמליזציה, לקבוע כוח terminator ביחס באמצעות המשוואה <img alt="משוואה 2" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

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The authors have nothing to disclose.

הצעד החשוב ביותר בפרוטוקול זה הוא עיצוב oligonucleotide הנכון לפני ההזמנה. Oligonucleotides חייבת להיות הקצוות הדביקים המתאימים הוסיפו אל קצוות &quot;5 של הן בשורה העליונה והן גדילים תחתונים כדי להבטיח כי התאגדות GGA אפשרית. בנוסף, חשוב לעבור את הכיוון של terminators מול שמאלה (terminators לזה לעצ?...

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can be completed in a relatively short time frame. 
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced. 
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.

<table><tbody><tr><td>pGR-Blue Plasmid</td><td>Addgene</td><td>68374</td><td></td></tr><tr><td>pGR-Plasmid</td><td>Addgene</td><td>46002</td><td></td></tr><tr><td>AeraSeal-(Sterile Sheets)</td><td>Excel Scientific</td><td>BS-25</td><td>Sterile Sheets only</td></tr><tr><td>10x T4 DNA ligase Buffer</td><td>NEB</td><td></td><td></td></tr><tr><td>BsaI-HF</td><td>NEB</td><td>R3535S</td><td>The non-HF enzyme will work but is less heat stable.&amp;nbsp;</td></tr><tr><td>NEB Golden Gate Assembly Mix</td><td>NEB</td><td>E1600S</td><td>Commerial Master Mix refered to in the protocol.</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Round Microcentrifuge Floating Rack</td><td>Nova Tech International</td><td>F18875-6401</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A9518</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-(+)-Arabinose</td><td>Sigma Aldrich</td><td>A-3256</td><td>D-Arabinose will not induce the pBAD promoter</td></tr><tr><td>Luria Base (LB) - Broth, Miller</td><td>Sigma Aldrich</td><td>L1900</td><td></td></tr><tr><td>Luria Base (LB) - Agar, Miller</td><td>Sigma Aldrich</td><td>L2025</td><td></td></tr><tr><td>Tecan-Infinite M200 Plate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Mix &amp;amp; Go Competent Cells - Strain JM109</td><td>Zymo Research</td><td>T3005</td><td>Use company recommended transformation protocol</td></tr><tr><td>ApE: A plasmid editor-software</td><td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td><td></td><td></td></tr><tr><td>Tris-HCl, Molecular Grade</td><td>Promega</td><td>H5121</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Biological, Certified)</td><td>Fisher Chemical</td><td>S671</td><td></td></tr><tr><td>Comercial Oligonucleotide synthesis</td><td>Integrated DNA Technologies (IDT)</td><td></td><td>http://www.idtdna.com/site</td></tr><tr><td>Microtest Tissue Culture Plates - 96 well (Sterile)</td><td>Falcon</td><td>35-3072</td><td></td></tr><tr><td>mycobacteriophage &quot;Bernal13&quot;</td><td>Genebank</td><td>KJ510413</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Integrated DNA Technologies (IDT)</td><td>IDT004</td><td></td></tr><tr><td>Sterile, L-shaped Hockey-Stick Cell</td><td>Life Science Products</td><td>6444-S1</td><td></td></tr><tr><td>Nano-Drop 2000c UV-Vis Spectrometer</td><td>Thermo Scientific</td><td>2000c</td><td></td></tr><tr><td>ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td><td>http://rna.igmors.u-psud.fr/</td><td></td><td></td></tr></tbody></table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

פרוטוקול זה יהיה לייצר תאים המכילים PGR-כחול עם terminator ligated בין ה- GFP ו RFP באמצעות שער זהב עצרת (איור 2). מושבות חיוביות המכילות כנס ligated ניתן לבחור על בסיס צבע. באור נראה מושבות חיוביות תהיינה לבנות / צהובות חיוביים שגוי תפקנה מושבות כחולות לאחר 18-...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

אימות של Terminators ראפיד שימוש פלסמיד PGR-כחול גולדן גייט האסיפה

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Research

JoVE Journal

Biology

7.6K Views.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

Video: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

מבחני מזווג Conjugative עבור ניתוח רצף ספציפי של חלבוני העברה מעורבים קוניוגציה

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

ניתוח של הפסקת תמלול שימוש BrUTP גדיל ספציפי התמלול Run-על (TRO) גישה

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

פרוטוקול אוניברסלי עבור gRNA בקנה מידה גדול ספריית הפקה מכל מקור ה-DNA

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

מסך 2-היברידית שמרים אצווה כדי להשוות בין אינטראקציות חלבון

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, תאים ייחודיים לסוג ביטוי תודרנית לבנה בתיווך דרך LhGR טרנס-הפעלה

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

הרכבה מהירה של מבנים מרובי גנים באמצעות שיבוט שער הזהב מודולרי

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengineering

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

מעגלים דיגיטליים גנטיים המבוססים על מערכות CRISPR-Cas וחלבונים אנטי-קריספר

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

עכבר הגנום הנדסה באמצעות nucleases מעצב

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

דור של פלסמיד וקטורים לבטא חלבונים מתויג FLAG על פי תקנות ההסדרה של יזם אדם התארכות Factor-1α שימוש גיבסון עצרת

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

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.

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 ...