עבודה זו מומנה על ידי גורדון ובטי מור קרן, ועל ידי היוזמה המדע UNCF / מרק.

1. צלחת הקרנת הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף LB 25 גרם צמיחה בינוני, אגר 15g ו 1.5g carboxymethyl תאית (CMC) המצע במים מזוקקים עד 1 ליטר ו החיטוי לעקר. </li><li style=";text-align:right;direction:rtl"> לאחר מעוקר, לאפשר בינוני להצטנן ולהוסיף אנטיביוטיקה המתאימה IPTG לריכוז סופי של 100μM. </li><li style=";text-align:right;direction:rtl"> יוצקים לתוך כל אחד בינוני 200 מ&quot;ל 5 צלחות פטרי גדול מרובע / מגשים המבדק (240 x 240 x 20 מ&quot;מ או יותר). אפשר צלחות להתייבש לחלוטין. </li></ol> 2. Endoglucanase הדור ספריה מסך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יצירת ספרייה של תחום קטליטי endoglucanase (ים). ניתן להשיג בדרכים רבות (למשל, אתר מכוונת או mutagenesis רקומבינציה אקראית, וכו &#39;) נקבעת על ידי החוקר. </li><li style=";text-align:right;direction:rtl"> Clone הספרייה לתוך וקטור כזה כי הם תחת שליטתה של האמרגן T7 עם המפעיל לאק, ומכילים רצף האות pelB עבור המיקוד periplasm. דוגמה וקטור מתאים pET22b (+) מ Novagen. </li><li style=";text-align:right;direction:rtl"> המרה הספרייה endoglucanase לתוך E. coli זן BL21 (DE3) ו צלחת מושבות אחת. </li><li style=";text-align:right;direction:rtl"> דגירה שינוי בן לילה ב 37 ° C. </li><li style=";text-align:right;direction:rtl"> בעזרת קיסמים סטרילי, לחסן שיבוטים לתוך 96 צלחות היטב היטב עמוק המכיל מדיום LB 400μL בתוספת אנטיביוטיקה מתאימה. לדגור על 37 מעלות צלזיוס לילה עם רועד בסל&quot;ד 250. </li><li style=";text-align:right;direction:rtl"> הוסף גליצרול סטרילי עד 96 תרבויות לילה טוב לריכוז סופי של 10% עבור אחסון לטווח ארוך ב -80 ° C. זו תהיה צלחת מאסטר. </li><li style=";text-align:right;direction:rtl"> באמצעות חותמת 96 פינים, לשכפל תרבויות צלחת לילה על גבי צלחות ההקרנה המכיל IPTG ו CMC. עד 5 סטים של תרבויות לילה יכול להיות מוטבע על כל צלחת ההקרנה. </li><li style=";text-align:right;direction:rtl"> לייבל צלחות ההקרנה כך זהות והכיוון של הצלחת 96 כל טוב ידוע. </li><li style=";text-align:right;direction:rtl"> דגירה הספריות חותמת בטמפרטורת החדר (17-25 מעלות) למשך הלילה או עד מושבות גלוי יצרו. </li><li style=";text-align:right;direction:rtl"> לשטוף צלחת עם מים מזוקקים עד כל עקבות המושבות יוסרו. </li><li style=";text-align:right;direction:rtl"> יוצקים קונגו האדום (CR; 0.5% במים) לצלחת שטף. הקפד להוסיף CR מספיק כדי לכסות את הצלחת. </li><li style=";text-align:right;direction:rtl"> דגירה 15 דקות בטמפרטורת החדר. לא יעלה על 15 דקות הדגירה CR. פעמים הדגירה ארוכים תגרום הילות פחות ברורים. </li><li style=";text-align:right;direction:rtl"> יוצקים את CR ומוסיפים כמות נדיבה (יותר ממספיק כדי לכסות את צלחת) של NaCl 1M. </li><li style=";text-align:right;direction:rtl"> דגירה 15 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> יוצקים את NaCl לחשוף הילות השפלה CMC. לקבלת רזולוציה טובה יותר של הילות, פעמים הדגירה כבר עם מכבסת NaCl NaCl או מרובים ייתכן שתידרש. </li><li style=";text-align:right;direction:rtl"> בעזרת קיסם סטרילי, להרים פעיל שיבוטים אנזימים מהצלחת האב אפיון נוסף. </li></ol> 3. נציג תוצאות: דוגמה readout עבור תפוקה גבוהה במסך זה מוצג באיור 3. המשובטים ניתן לזהות פעיל, פעיל חלושות, ופעיל מאוד בהתבסס על גודל של אזורי השפלה. תכונה זו שימושית במיוחד אם אנזים הפעילות ידוע נכלל בספריה כהפניה. כפי שמוצג כאן, זיהוי של אנזימים פעילים איתנה לשחזור. עם זאת, יש להיות מודעים לכך תיוג נאותה של לוחות ההקרנה הוא מאוד חשוב. החוקר חייב להיות מסוגל לזהות גרסאות פעיל לאחר כביסה המושבות משם, על מנת לאסוף אותם מהצלחת האב מאוחסנים -80 ° C לאפיון נוסף. לבסוף, קשה להעריך מראש את הקשר בין פעילות על מצעים מלאכותיים מול טבעיים. לכן, החוקר חייב לבדוק את כל שיבוטים חיובית על חומר תעשייתי רלוונטי לקביעת כושר חלבון. <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="איור 1"/> באיור 1. מתווה סכמטי של תהליך המיון. תאים הופכים עם ספריה endoglucanase ו מצופה סלקטיבי עבור מושבות אחת. המשובטים הרים גדל בן לילה לאחסון (1) ב -80 ° C ו (2) ציפוי העתק על צלחות המכילות CMC ו IPTG לגרום ביטוי endoglucanase ב 96 צלחות היטב עמוק היטב. שיבוטים פעילים נקטפים מהצלחת האב מאוחסנים -80 ° C. <img src="/files/ftp_upload/2942/2942fig2.jpg" alt="איור 2"/> איור 2. שיבוט וביטוי של תחומים קטליטי פטרייתי ב E. coli. (א) וקטור Endoglucanase הביטוי. הגנים היו משובטים לתוך pET22b (+) (Novagen) תחת שליטה של ​​האמרגן T7. (ב) פאנל ימין: endoglucanase-לבטא BL21 (DE3) המושבות. פאנל משמאל: הילות השפלה CMC לאחר שטיפה ופיתוח עם קונגו האדום. <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="איור 3"/> איור 3. תוצאות המדגם מן המסך האדום CMC / קונגו. תמונות שצולמו של צלחות ההקרנה לאחר שטיפה ופיתוח קונגו האדום. כל מושבות על זהצלחות creening היו בסדר גודל דומה.

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Macrophomina+phaseolina+endoglucanase+(EGL+1)+gene+product+in+bacterial+and+yeast+expression+systems.">Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.</a> Appl Biochem Biotechnol. 81, 153-160 (1999).</li><li>Howard, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+activity+of+a+Phanerochaete+chrysosporium+cellobiohydrolase+(CBHI.1)+expressed+as+a+heterologous+protein+from+Escherichia+coli.">Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli.</a> Afr J Biotechnol. 2, 296-300 (2003).</li><li>Howard, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+a+chimeric+Phanerochaete+chrysosporium+cellobiohydrolase+expressed+from+Escherichia+coli.">Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli.</a> Afr J Biotechnol. 3, 349-352 (2004).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+red-polysaccharide+interactions+in+enumeration+and+characterization+of+cellulolytic+bacteria+from+the+bovine+rumen.">Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen.</a> Appl Env Microbiol. 43, 777-780 (1982).</li><li>Gilkes, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+action+and+substrate+specificities+of+cellulases+from+cloned+bacterial+genes.">Mode of action and substrate specificities of cellulases from cloned bacterial genes.</a> J Bio Chem. 259, 10455-10459 (1984).</li></ol>

אין ניגודי אינטרסים הכריז.

פרוטוקול המתואר כאן מאפשר זיהוי מהיר תפוקה גבוהה של אנזימים פעילים, עם מניפולציה מינימלית. איתור פעילות רגיש למדי, איכותית משקף את היקף הפעילות בתוך התא. קלות השימוש שעושה שיטה זו מתאימה למגוון רחב של ספריות האנזים, מוגבל רק על ידי ביטוי פונקציונלית E. coli. יתר על כ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> מגיב </th><th> חברה </th><th> מספר מוצר </th></tr><tr><td> IPTG </td><td> סיגמא </td><td> I1284 </td></tr><tr><td> קונגו אדום </td><td> סיגמא </td><td> C6277 </td></tr><tr><td> Carboxymethyl תאית </td><td> סיגמא </td><td> 360384 </td></tr><tr><td> NaCl </td><td> סיגמא </td><td> S1679 </td></tr><tr><td> Bacto-agar </td><td> BD Biosciences </td><td> 214030 </td></tr><tr><td> המבדק צלחות </td><td> Thermo Scientific </td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

Cellulase אנזימים (endoglucanases, cellobiohydrolases, ו β-glucosidases) hydrolyze תאית לסוכרים רכיב, אשר בתורו יכול להיות מומר כהלים דלק 1. הפוטנציאל הידרוליזה אנזימטית של ביומסה cellulosic לספק אנרגיה מתחדשת הגביר את המאמצים מהנדס cellulases עבור 2 דלק חסכוני הייצור. עניין מיוחד הם cellulases פטרייתי 3-8, אשר כבר נמצאים בשימוש תעשייתי עבור מזונות ועיבוד טקסטיל. זיהוי וריאנטים פעיל בין ספריה של cellulases מוטציה הוא קריטי לתהליך ההנדסה; מוטנטים פעילים ניתן לבדוק נוספת תכונות משופרות ו / או נתון mutagenesis נוספים. הנדסה יעיל של cellulases פטרייתי כבר הקשו על ידי היעדר כלים גנטית אורגניזמים מקומיים על ידי קשיים בהבעת האנזימים המארחים Heterologous. לאחרונה, פיתח Morikawa ועמיתיו שיטה לביטוי ב E. coli התחומים הקטליטית של endoglucanases מ ח jecorina 3,9, פטריות תעשייתי חשוב עם יכולת להפריש cellulases בכמויות גדולות. פונקציונלית E. הביטוי coli כבר דווח על cellulases מן פטריות אחרים, כולל Macrophomina phaseolina 10 ו Phanerochaete chrysosporium 11-12. אנו מציגים שיטה ההקרנה תפוקה גבוהה של פעילות endoglucanase פטרייתי ב E. coli. (איור 1) שיטה זו משתמשת חיידקים המשותף צבען האדום קונגו (CR) כדי להמחיש השפלה אנזימטית של תאית carboxymethyl (CMC) על ידי תאים גוברת על בינוני מוצק. Assay פעילות דורשת ריאגנטים זולה, מניפולציה מינימלית, נותן תוצאות חד משמעיות כמו אזורי השפלה (&quot;הילות&quot;) במקום מושבה. למרות מדד כמותי של הפעילות האנזימטית לא ניתן לקבוע לפי שיטה זו, מצאנו כי גודל הילה בקורלציה עם הפעילות האנזימטית הכולל בתא. אפיון נוסף של שיבוטים חיובי הפרט יקבע, כושר חלבון יחסית. מסורתית חיידקי כל CMC / CR תא פעילות מבחני 13 לערב לשפוך CMC אגר המכיל על מושבות, אשר כפופה זיהום צולב, או תרבויות דוגרים בבארות אגר CMC, המהווה פחות נגישה ניסויים בקנה מידה גדול. כאן אנו מדווחים על פרוטוקול משופר משנה לשטוף שיטות קיימות 14 לפעילות cellulase: התאים גדלו על צלחות אגר CMC מוסרים לפני מכתים CR. פרוטוקול שלנו מפחית באופן משמעותי את זיהום צולבות היא מדרגית, המאפשר הקרנת מהירה של אלפי שיבוטים. בנוסף ח אנזימים jecorina, יש לנו הביע הוקרן וריאנטים endoglucanase מן aurantiacus Thermoascus ו Penicillium decumbens (שמוצג באיור 2), טוען כי פרוטוקול זה הוא ישים אנזימים מתוך מגוון רחב של אורגניזמים.

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

אנו מתארים עלות נמוכה, גבוה שיטת התפוקה למסך לפעילות endoglucanase פטרייתי ב<em&gt; א coli.</em&gt; השיטה מסתמכת על readout ויזואלית פשוטה של ​​השפלה המצע, אינו דורש טיהור האנזים, והיא ניתנת להרחבה. זה מאפשר הקרנה מהירה של ספריות גדולות של וריאנטים של האנזים.

הקרנת תפוקה גבוהה של פעילות Endoglucanase פטרייתיים ב Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

high-throughput-screening-fungal-endoglucanase-activity-escherichia

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.4K Views.  California Institute of Technology. אנו מתארים עלות נמוכה, גבוה שיטת התפוקה למסך לפעילות endoglucanase פטרייתי ב א coli. השיטה מסתמכת על readout ויזואלית פשוטה של ​​השפלה המצע, אינו דורש טיהור האנזים, והיא ניתנת להרחבה. זה מאפשר הקרנה מהירה של ספריות גדולות של וריאנטים של האנזים.

Video: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Assay למדידת פעילות Escherichia coli ועין מתנהלת ליזין Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

מסך התפוקה גבוהה עבור Biomining פעילות cellulase מספריות Metagenomic

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

תפוקה גבוהה הקרנת ו biosensing עם פלורסנט ג elegans מתחים

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

הקרנת תפוקה גבוהה למאפנני מולקולה הקטנה של ערוצי אשלגן מיישרים פנימה

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

הקרנה פונקציונלית תפוקה גבוהה באמצעות assay בלוציפראז Dual-זוהר תוצרת בית

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

הקרנת תפוקה גבוהה ביטוי כמותי וטיהור יישומים לחלבוני ארס רקומביננטי דיסולפיד עשירים הופק ב<em&gt; א coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

הקרנה ירוקה פלורסנט מבוסס חלבון ביטוי של החלבונים ממברנה ב<em&gt; Coli Escherichia</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

תועלת רבה פנים של Co-ביטוי חלבון ב<em&gt; Coli Escherichia</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

הקרנת תפוקה גבוהה עבור כימית מאפננים של גני Post-תעתיק מוסדר

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

זיהוי של זוגות קינאז-מצע שימוש הקרנת תפוקה גבוהה


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...