Name: single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)
Uploaded: 2013-03-15T12:00:00
Description: Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Plasmidet pCG001 udtrykker UBP1 blev venligst stillet til rådighed af Rohan Baker på John Curtin School of Medical Research. Dette arbejde blev finansieret af March of Dimes Research Grant 1-FY2011, March of Dimes Basil O&#39;Connor Starter Scholar Research Award # 5-FY20 og NSF KARRIERE prisen 0.746.796.

1. Stamme Engineering Workflow <ol><li> Indsæt sekvenser der koder for (a) et membran-lokaliseringssekvens, (b) en hurtigt modning fluorescerende protein og (c) en proteasegenkendelsessekvens N-terminalt for og i ramme med et protein af interesse (fx en transkriptionsfaktor). Vi anvendte membranen målrettet TSR-Venus reporter 2 og fusioneres den til proteasegenkendelsessekvens Ubiquitin (Ub) til at tælle antallet af udtrykte bakteriofag λ repressor CI proteinmolekyler. Detaljer om, hvordan vi k...

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	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., 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=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

The CoTrAC fremgangsmåde kan generaliseres til at måle produktionen af ​​andre proteiner, hvor konventionelle N-eller C-terminale fluorescerende proteinfusioner kan forstyrre proteinaktivitet. Den CoTrAC strategi har tre unikke fordele i forhold til de nuværende metoder. Første, co-translationel fusion sikrer, at et molekyle af det fluorescerende reporter fremstilles for hvert molekyle af proteinet af interesse, hvilket tillader nøjagtig optælling af proteinproduktion i realtid. For det andet har membranen må...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Agarose (lav smeltetemperatur) </td><td> Lonza </td><td> 50.100 </td><td></td></tr><tr><td> Milli-Q H2O </td><td></td><td></td><td></td></tr><tr><td> 5 x M9-salte </td><td></td><td></td><td> Følgende opskrift beskrevet i 9 </td></tr><tr><td> 20% glucose </td><td></td><td></td><td></td></tr><tr><td> MgSO4 </td><td></td><td></td><td></td></tr><tr><td> CaCl2 </td><td></td><td></td><td></td></tr><tr><td> 50 × MEM aminosyreopløsning </td><td> Invitrogen </td><td> 11130-051 </td><td></td></tr><tr><td> Temperatur-kontrolleret vækstChamber Stage adapter </td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td> Mål Heater </td><td> Bioptechs </td><td> Model afhænger mikroskopobjektiv </td><td></td></tr><tr><td> Microaqueduct Slide </td><td> Bioptechs </td><td> 130.119-5 </td><td></td></tr><tr><td> Micro dækglasset </td><td> VWR </td><td> 40CIR-1 </td><td> Kan være vanskeligt at kilden også tilgængelig fra Bioptechs </td></tr><tr><td> Cover glas / slide pakning </td><td> Bioptechs </td><td> FCS2 0,75 mm </td><td></td></tr><tr><td> Fluorescensmikroskop </td><td> Various </td><td> Eksempel setup: Sammenhængende Innova 308c Argon-ion laser, Olympus IX-81-mikroskop, Olympus PlanApo 100X NA 1,45 mål, Metamorph software </td><td> Skal have laser excitation, automatiseret xyz scene, automatiserings-software er i stand til scriptede billedbehandling og autofokus, optik kan resolving enkelte fluorescerende proteiner </td></tr><tr><td> EM-CCD-kamera </td><td> Various </td><td> Eksempel opsætning: Andor Ixon DU-898 </td><td> Skal have tilstrækkelig lav støj til påvisning af enkelte fluorescerende proteiner over baggrunden </td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

Vi beskriver en fluorescensmikroskopi fremgangsmåde, cotranslationel aktivering ved spaltning (CoTrAC) til billede produktionen af ​​proteinmolekyler i levende celler med enkelt-molekyle præcision uden at forskubbe proteinets funktionalitet. Denne metode gør det muligt at tælle antallet af proteinmolekyler produceret i én celle i sekventielle, fem minutter tidsvinduer. Det kræver et fluorescensmikroskop med laserexcitation effekttæthed på ~ 0,5 til 1 kW / cm 2, som er tilstrækkelig følsom til at påvise enkelte fluorescerende protein-molekyler i levende celler. Det fluorescerende reporter, der anvendes i denne fremgangsmåde består af tre dele: en membran targeting sekvens, en fast-modning, gult fluorescerende protein og en protease-genkendelsessekvens. Reporteren er translationelt fusioneret til N-terminalen af ​​et protein af interesse. Celler dyrkes på en temperaturstyret objektbordet. Hvert femte minut, er fluorescerende molekyler i celler afbildes (og senere counted ved at analysere fluorescensbilleder) og efterfølgende Lysblegede således at kun nyligt translaterede proteiner tælles i den næste måling. Fluorescensbilleder som følge af denne fremgangsmåde kan analyseres ved at detektere fluorescerende pletter i hvert billede, tildele dem til individuelle celler og derefter tildele celler til cellelinier. Antallet af proteiner i et tidsvindue i en given celle beregnes ved at dividere den integrerede fluorescensintensitet af pletter med den gennemsnitlige intensitet af enkelte fluorescerende molekyler. Vi anvendte denne fremgangsmåde til at måle ekspressionsniveauer i området fra 0 til 45 molekyler i enkelt-5 min tidsvinduer. Denne fremgangsmåde gjorde det muligt at måle støjen i ekspressionen af ​​λ-repressoren CI, og har mange andre potentielle anvendelser i systemer biologi.

Typiske resultater fra en CoTrAC eksperiment sporing produktionen af λ-repressoren CI er vist i figur 4 og 5.. I dette forsøg blev 12 kolonier afbildet ved 5 minutters intervaller. På hvert tidspunkt blev kolonien 1:e autofocused og centraliseret i imaging-regionen. Dernæst blev centrerede / fokuserede position lagres og en brightfield billede blev taget. Etapen blev derefter translokeret ved ~ 0,5 um langs z-aksen til at flytte fra den brightfield brændplan til flyet skærer cellerne (uden fase-ko...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Vi beskriver en fluorescensmikroskopi fremgangsmåde, cotranslationel aktivering ved spaltning (CoTrAC), til billede produktionen af ​​proteinmolekyler i levende celler med enkelt-molekyle præcision uden at forskubbe proteinets funktionalitet. Denne fremgangsmåde er blevet anvendt til at følge de stokastiske udtrykket dynamik en transkriptionsfaktor, at λ repressor CI<sup&gt; 1</sup&gt;.

Single-molekyle Imaging af genregulering<em&gt; In vivo</em&gt; Brug Cotranslational Aktivering ved spaltning (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

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Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

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