Name: a yeast 2-hybrid screen in batch to compare protein interactions
Uploaded: 2018-06-06T14:30:00
Description: Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

Vi takker personale inden for Institut for humangenetik for NGS bibliotek forberedelse og sekventering. Vi takker Einat Snir for hendes ekspertise i forberede Y2H plasmid biblioteket her genomiske biblioteket fragmenter. Dette arbejde blev støttet af National Institutes of Health: NIH R21 EB021870-01A1 og af NSF Research Project Grant: 1517110.

1. forberedelse af medier og plader
Bemærk: Alle plader skal foretages minimalt 2 dage før begyndelsen af protokollen. Medierne kan foretages på ethvert tidspunkt. Bufferet gær extract pepton dextrose adenin (bYPDA) skal dog ske den dag, hvor det vil blive brugt. Nogle medier er lavet ved hjælp af et supplement mix som indeholder et niveau af adenin, der er større end hvad der typisk bruges. De fleste minimal media kosttilskud angive 10 mg/L adenin. Kosttilskud mærket '+ 40Ade' Angiv ialt...

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Bruckner, A., Polge, C., Lentze, N., Auerbach, D., Schlattner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+two-hybrid,+a+powerful+tool+for+systems+biology.">Yeast two-hybrid, a powerful tool for systems biology.</a> International Journal of Molecular Sciences. 10 (6), 2763-2788 (2009).</li><li>Vidal, M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+two-hybrid+assay:+still+finding+connections+after+25+years.">The yeast two-hybrid assay: still finding connections after 25 years.</a> Nature Methods. 11 (12), 1203-1206 (2014).</li><li>Pashkova, N., 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=DEEPN+as+an+Approach+for+Batch+Processing+of+Yeast+2-Hybrid+Interactions.">DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.</a> Cell Reports. 17 (1), 303-315 (2016).</li><li>Rajagopala, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens.">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li><li>Weimann, M., 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+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome.">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li><li>Yachie, N., 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=Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics.">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863 (2016).</li><li>Trigg, S. A., 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=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. T., Uetz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+yeast+two-hybrid+systems+using+the+interactions+of+bacterial+motility+proteins.">Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.</a> Proteomics. 9 (23), 5296-5302 (2009).</li><li>James, P., Halladay, J., Craig, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+libraries+and+a+host+strain+designed+for+highly+efficient+two-hybrid+selection+in+yeast.">Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.</a> Genetics. 144 (4), 1425-1436 (1996).</li><li>Barbas, C. F., Burton, D. R., Scott, J. K., Silverman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+and+RNA.">Quantitation of DNA and RNA.</a> CSH Protoc. 2007, pdb ip47 (2007).</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=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. 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Her giver vi en guide til hvordan man udfører Y2H assays i batch optimerede metoder. Der er et par kritiske trin i proceduren for at sikre, at befolkningen af gær, der ville være placeret under valg er repræsentant for biblioteket start og, nok af befolkningens start gær bruges til at gennemgå udvalg for at begrænse variabilitet . Vigtigere, er disse benchmarks relativt let at opnå sammen med tilpasning af metoder og materialer til en traditionel Y2H, analyse, således at denne tilgang gøres tilgængelige for de...

En komplet forståelse af celle biologiske processer er baseret på at finde de protein interaktion netværk, der ligger til grund for de molekylære mekanismer. En metode til at identificere protein interaktioner er gær 2-hybrid (Y2H) analyse, som virker ved at samle en fungerende kimære transkriptionsfaktor, når to protein domæner af interesse binde til hinanden1. En typisk Y2H skærmen er udført ved at skabe en befolkning af gær, der huser både et bibliotek af plasmider kodning interagerende proteiner smeltet til en transcriptional aktivator (fx., 'bytte' fusion protein) og en given 'agn' plasmid består af protein af interesse er sammenvoksede til en DNA bindende domæne (f.eks., Gal4 DNA-bindende domæne, der binder til den Gal4-upstream aktiverende sekvens). En af de største fordele ved Y2H tilgangen er at det er relativt nemt og billigt at foretage i en typisk laboratorium udstyret for Molekylær biologiske rutinearbejde2. Men når traditionelt udført, en bruger prøver enkelte kolonier, der opstår efter valget for en positiv Y2H interaktion. Dette begrænser i høj grad antallet af bibliotek 'bytte' kloner, der kan blive undersøgt. Dette problem forværres, når overfloden af en bestemt interagerende bytte er meget høj i forhold til de andre, mindre chance for at opdage interaktion fra lav overflod bytte plasmider.
En løsning for at bruge Y2H princippet i omfattende dækning af proteomet er brugen af en matrix-formateret tilgang, hvori et array som indeholder kendte individuelle bytte plasmider kan være digitalt afhørt. Men en sådan tilgang kræver en infrastruktur, der ikke er umiddelbart tilgængelig eller omkostningseffektivt at enkelte undersøgelsesmedarbejdere, der er interesseret i at definere interactome af et lille antal proteiner eller domæner3. Derudover vil meget komplekse bytte biblioteker, der kan indkode flere fragmenter af interagerende proteiner udvide størrelsen af sådanne matrix arrays til upraktisk størrelser. Et alternativ er at udføre assays med komplekse biblioteker i partier og vurdere tilstedeværelsen af interagerende kloner ved hjælp af massive parallelle høj overførselshastighed sekventering4. Dette kan anvendes til at assay tilstedeværelsen af bytte plasmider, der opstår i flere kolonier ved hjælp af en typisk Y2H formateret tilgang i hvilken gær celler boliger en interagerende par fusion proteiner får lov til at vokse på en plade5,6. Denne generelle idé kan blive forstærket for at øge forespørgsel af begge flere agn og bytte komponenter på samme tid7,8.
Stadig, mange undersøgelser kræver en lettere endnu mere fokuseret indsats på blot et par protein "lokkemad" og kan drage større fordel af en udtømmende og semi-kvantitative forespørgsel om en enkelt kompleks bytte bibliotek. Vi har udviklet og valideret en metode for at udføre omfattende protein interaktion undersøgelser ved hjælp af et Y2H princip i batch format4. Dette bruger satsen for udvidelse af en bestemt bytte plasmid som en proxy for den relative styrke af Y2H interaktion9. Dyb sekventering af alle plasmider inden for en befolkning udsættes for normal vækst eller selektiv vækstbetingelser producerer et komplet kort over kloner, der giver stærke og svage Y2H interaktioner. Repertoire af interactors kan opnås og direkte sammenlignes på tværs af flere agn plasmider. Den resulterende arbejdsproces kaldes DEEPN (dynamisk berigelse for evaluering af Protein netværk) kan således bruges til at identificere differentieret interactomes fra de samme byttedyr biblioteker til at identificere proteiner, tillader sammenligning mellem et protein vs en anden.
Her, vi demonstrere DEEPN og indføre forbedringer i de laboratoriemetoder, der letter brugen, som er skitseret i figur 1. Betydelige forbedringer omfatter:
Generation af bytte gær populationer. En af de vigtigste krav til DEEPN genererer populationer af gær med forskellige agn plasmider, der har den samme fordeling af plasmid bytte biblioteker. Tilsvarende oprindelige befolkninger af bytte plasmid bibliotek er afgørende for at gøre nøjagtig sammenligninger mellem interactomes af forskellige lokkemad. Dette opnås bedst, når en bibliotek plasmid allerede har til huse i en haploid gær befolkning og flytter en given agn plasmid i befolkningen er opnået ved parring for at producere en diploid. Her give vi en klar vejledning i hvordan man laver disse befolkninger ved hjælp af kommercielle biblioteker har til huse i haploide gær. Selv om vi fundet metoder, der genererer et stort antal diploids, var den samlede parring effektivitet af disse kommercielle bibliotek, der indeholder gærstammer lav. Derfor, vi konstrueret en ny stamme, der kan huse bytte biblioteker, der giver langt flere diploids per parring reaktion.
Nye sæt af agn plasmider. Mange nuværende plasmider, der udtrykker 'agn' fusion proteiner består af protein af interesse og en DNA-bindende domæne er baseret på 2µ, får mulighed at forstærke deres kopi nummer. Denne kopi antallet kan være meget varierende i befolkningen og føre til variabilitet i Y2H transcriptional svar. Dette kunne til gengæld skew evnen til at måle styrken af et bestemt protein interaktion baseret på vækst svar af celler under udvælgelse. Dette kan være delvis adresse ved hjælp af en lav kopi plasmid, hvoraf nogle har været tidligere beskrevet som kommercielt tilgængelig pDEST3210. Vi konstrueret en ny agn plasmid (pTEF-GBD), der producerer Gal4-DNA-bindende domæne fusion proteiner inden for en TRP1 centromer-baserede lav kopi plasmid bærer Kanr resistens gen, der også tillader kloning af agn fragmenter, både opstrøms og neden for Gal4 DNA-bindende domæne.
Nye High-Density Y2H fragment bibliotek. Vi konstrueret en ny plasmid til huset Y2H bytte biblioteker og brugte det til at bygge en yderst komplekse Y2H bibliotek lavet af tilfældigt afrevne fragmenter af genomisk DNA fra Saccharomyces cerevisiae. Sekvensanalyse viste, at biblioteket havde over 1 million forskellige elementer, langt mere kompleks end tidligere beskrevet gær genomisk Y2H plasmid biblioteker11. Med denne nye bibliotek har vi kunnet vise, at DEEPN arbejdsproces er robuste nok til at rumme komplekse biblioteker med mange forskellige plasmider på en måde, der er pålidelig og reproducerbar.

<table>
	<tbody>
		<tr>
			<td>Illumina HiSeq 4000</td>
			<td>Illumina</td>
			<td></td>
			<td>deep sequencing platform</td>
		</tr>
		<tr>
			<td>Monoclonal anti-HA antibodies</td>
			<td>Biolegend</td>
			<td>901514</td>
			<td>Primary Antibody to detect expression of HA in pGal4AD constructs</td>
		</tr>
		<tr>
			<td>Polyclonal anti-myc antibodies</td>
			<td>QED Biosciences Inc</td>
			<td>18826</td>
			<td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td>
		</tr>
		<tr>
			<td>NarI</td>
			<td>New England BioLabs</td>
			<td>R0191S</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England BioLabs</td>
			<td>R3236S</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England BioLabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 3350, powder</td>
			<td>J.T. Baker</td>
			<td>U2211-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Trevigen, Inc sold by Fisher Scientific</td>
			<td>50-948-286</td>
			<td>carrier DNA for yeast transformation section 3.2.1.</td>
		</tr>
		<tr>
			<td>Kanamycin Monosulfate</td>
			<td>Research Products International</td>
			<td>K22000</td>
			<td></td>
		</tr>
		<tr>
			<td>LE Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td>used for making DNA agarose gels</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Research Products International</td>
			<td>S23025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Research Products International</td>
			<td>T60060</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Research Products International</td>
			<td>S23080</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>MP Biomedicals</td>
			<td>155256</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Sodium Salt</td>
			<td>Research Products International</td>
			<td>E57020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Powder</td>
			<td>Research Products International</td>
			<td>Y20020</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td>
			<td>Research Products International</td>
			<td>Y20040</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu+40ADE</td>
			<td>Formedium</td>
			<td>DCS0789</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Leu-His+40ADE</td>
			<td>Formedium</td>
			<td>DCS1169</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu-Met</td>
			<td>Formedium</td>
			<td>DCS0549</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Trp-Met</td>
			<td>Bio 101, Inc</td>
			<td>4520-922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Formedium</td>
			<td>DOC0168</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Research Products International</td>
			<td>A11500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Research Products International</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>used for making media plates in section 1</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Research Products International</td>
			<td>P20240</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino-1,2,4 Triazole</td>
			<td>Sigma</td>
			<td>A8056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoehanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>USBiological</td>
			<td>Z1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma</td>
			<td>P8281</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA</td>
			<td>Ambion</td>
			<td>AM9732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>238074</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories, INC</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>ThermoFisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Research Products International</td>
			<td>U20200</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Research Products International</td>
			<td>L22010</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Gibco</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Amresco</td>
			<td>449</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Cloning Kit</td>
			<td>New England Biolabs</td>
			<td>E5510S</td>
			<td>Rapid assembly method for cloning of plasmids in section 2</td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0541S</td>
			<td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>0492-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Used for purification of pcr products in section 6.2.3</td>
		</tr>
		<tr>
			<td>Qiaquick DNA Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Used for purification of digested pTEF-GBD in section 2.1</td>
		</tr>
		<tr>
			<td>KAPA Hyper Prep kit</td>
			<td>KAPA Biosystems</td>
			<td>KK8500</td>
			<td>preparation kit for deep sequencing</td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>http://www.jcat.de</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Codon optimization</td>
			<td>https://www.idtdna.com/CodonOpt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gBlocks</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>Strings</td>
			<td>Thermofisher</td>
			<td></td>
			<td>DNA fragments used for cloning in Section 2.2</td>
		</tr>
		<tr>
			<td>GenCatch Plasmid DNA mini-prep Kit</td>
			<td>EPOCH Life Sciences</td>
			<td></td>
			<td>Used to prepare quantities of DNA in Section 2.3</td>
		</tr>
		<tr>
			<td>Covaris E220</td>
			<td>Covaris</td>
			<td></td>
			<td>high performance ultra-sonicator in section 7</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;- CGGTCTT 
			CAATTTCTCAAGTTTCAG -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GAGTAACG 
			ACATTCCCAGTTGTTC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 5&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-CACCGTAT 
			TTCTGCCACCTCTTCC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligo nucelotide 5&#8217;-GCAACCGC 
			ACTATTTGGAGCGCTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>used for pcr amplification and sequencing 3&#39; insert pTEF-GBD during plasmid construction</td>
		</tr>
		<tr>
			<td>oligonucleotide 5&#8217;-GTTCCGATG 
			CCTCTGCGAGTG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-GCACATGCT 
			AGCGTCAAATACC-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGAL4AD</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;-ACCCAAGCA 
			GTGGTATCAACG-3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>5&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>oligonucelotide 5&#8217;- TATTTAGA 
			AGTGTCAACAACGTA -3&#8217;</td>
			<td>Integrated DNA Technologies or Thermofisher</td>
			<td></td>
			<td>3&#39; Pimer used for insert amplification of pGADT7</td>
		</tr>
		<tr>
			<td>PJ69-4A MatA yeast strain</td>
			<td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td>
			<td></td>
			<td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td>
		</tr>
		<tr>
			<td>pTEF-GBD</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-DNA binding doimain expression plasmid</td>
		</tr>
		<tr>
			<td>PLY5725 MATalpha yeast strain</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>MATalpha his3&#8710;1 leu2&#8710;0 lys2&#8710;0 ura3&#8710;0 gal4&#8710; trp1&#8710; Gal80&#8710;</td>
		</tr>
		<tr>
			<td>pGal4AD (pPL6343)</td>
			<td>Dr. Robert Piper Lab</td>
			<td></td>
			<td>Gal4-activation domain expression plasmid</td>
		</tr>
		<tr>
			<td>100 mm petri dishes</td>
			<td>Kord-Vallmark sold by VWR</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63270</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63271</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63274</td>
			<td></td>
		</tr>
		<tr>
			<td>2,000 mL PYREX Erlenmeyer flask</td>
			<td>Fisher Scientific</td>
			<td>S63275</td>
			<td></td>
		</tr>
		<tr>
			<td>20 X 150 mm Disposable Culture Tube</td>
			<td>Thermofisher</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>pipet-aid</td>
			<td>Drummond</td>
			<td>4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7128</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Serological Pipette</td>
			<td>Denville</td>
			<td>P7129</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Griffin Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-540P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL PYREX Reuasable Media Storage Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 mL graduated cylinder</td>
			<td>Fisher Scientific</td>
			<td>08-572-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax 190</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>used to measure the Optical Density of cells</td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Spectrophotometer used to quantify amount of DNA</td>
		</tr>
		<tr>
			<td>Electronic UV transilluminator</td>
			<td>Ultra Lum</td>
			<td>MEB 20</td>
			<td>used to visualize DNA in an Ethidium Bromide agarose gel</td>
		</tr>
		<tr>
			<td>P1000 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123602G</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123601G</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F123600G</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 Gilson PIPETMAN</td>
			<td>Fisher Scientific</td>
			<td>F144802G</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Low Retention Pipette Tips</td>
			<td>GeneMate</td>
			<td>P-1236-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;LLow Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-044</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L XL Low Retention Pipette Tips</td>
			<td>VWR</td>
			<td>10017-042</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>VWR</td>
			<td>490001-627</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>VWR</td>
			<td>490001-621</td>
			<td></td>
		</tr>
		<tr>
			<td>cell scraper</td>
			<td>Denville Scientific</td>
			<td>TC9310</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fluka Analytical</td>
			<td>318949-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Baker</td>
			<td>5674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wooden applicators</td>
			<td>Solon Care</td>
			<td>55900</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424</td>
			<td>Fisher Scientific</td>
			<td>05-400-005</td>
			<td>microcentrifuge</td>
		</tr>
		<tr>
			<td>Sorvall ST16R</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004381</td>
			<td>benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td>
			<td>GE Healthcare</td>
			<td>NA931-1ML</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34080</td>
			<td>ECL detection solution</td>
		</tr>
		<tr>
			<td>Isotemp Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Incubator</td>
		</tr>
		<tr>
			<td>Mutitron 2</td>
			<td>INFORS HT</td>
			<td></td>
			<td>Shaking incubator</td>
		</tr>
		<tr>
			<td>Isotemp Digital-Control Water Bath Model 205</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>water bath</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (pan tissue)</td>
			<td>Clontech</td>
			<td>630482</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>Y2H mouse cDNA library in Y187 (mouse brain)</td>
			<td>Clontech</td>
			<td>630488</td>
			<td>commercially available cDNA Library</td>
		</tr>
		<tr>
			<td>pGADT7 AD Vector</td>
			<td>Clontech</td>
			<td>630442</td>
			<td>commercially available AD Vector housing many cDNA libraries</td>
		</tr>
		<tr>
			<td>pGBKT7 DNA-BD Vector</td>
			<td>Clontech</td>
			<td>630443</td>
			<td>commercially available DNA-BD Vector</td>
		</tr>
		<tr>
			<td>Biolase DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21042</td>
			<td>DNA polymerase used for section 2.4</td>
		</tr>
		<tr>
			<td>GeneMate GCL-60 Thermal Cycler</td>
			<td>BioExpress</td>
			<td>P-6050-60</td>
			<td>pcr machine</td>
		</tr>
		<tr>
			<td>TempAssure 0.5 mL PCR tubes</td>
			<td>USA Scientific</td>
			<td>1405-8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a yeast 2-hybrid screen in batch to compare protein interactions

Screening for protein-protein interaktioner ved hjælp af gær 2-hybrid assay har længe været et effektivt redskab, men dets anvendelse har stort set været begrænset til opdagelsen af høj-affinitet interactors, der er stærkt beriget i biblioteket af interagerende kandidater. I en traditionel format, kan gær 2-hybrid assay give for mange kolonier til at analysere når udført på lav strenghed hvor lav affinitet interactors kan findes. Desuden, uden en omfattende og komplet forhør i det samme bibliotek mod forskellige agn plasmider, en sammenlignende analyse kan ikke opfyldes. Selv om nogle af disse problemer kan løses ved hjælp af klædt bytte biblioteker, kan omkostninger og infrastruktur forpligtet til at drive sådanne skærme være uoverkommelige. Som et alternativ, har vi tilpasset gær 2-hybrid assay for at samtidig afdække snesevis af forbigående og statisk protein interaktioner indenfor en enkelt skærm bruger en strategi kaldet DEEPN (dynamisk berigelse for evaluering af Protein netværk), som indeholder høj overførselshastighed DNA-sekventering og computation at følge udviklingen af en population af plasmider, der indkode interagerende partnere. Her beskriver vi tilpassede reagenser og protokoller, der giver mulighed for en DEEPN skærm skal udføres nemt og omkostningseffektivt.

Y2H analysen har været almindeligt brugt til at finde protein: protein interaktioner og flere tilpasninger og systemer er blevet udviklet. For det meste er de samme overvejelser, der hjælpe med at sikre succes med disse tidligere tilgange vigtige for DEEPN. Nogle af de vigtige referencepunkter omfatter: at sikre udtryk for DNA-bindende domæne fusion proteiner, sikre en lav baggrund af spurious hans + vækst i diploids som indeholder agn af interesse med en tom bytte plasmid, en parring...

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

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

En gær 2-Hybrid skærm i Batch for at sammenligne Protein interaktioner

Batchafvikling af gær 2-hybrid skærme giver mulighed for direkte sammenligning af interaktion profiler af flere agn proteiner med et meget komplekst sæt af bytte fusion proteiner. Her, beskriver vi raffineret metoder, nye reagenser, og hvordan man gennemfører deres brug for sådanne skærme.

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 ...

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Fluorescens Anisotropi som værktøj til at studere protein-protein interaktioner

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Brug Stillads Liposomer at Rekonstituer Lipid-proksimal protein-protein interaktioner<em&gt; In vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Time-løst elektrosprayioniserings Brint-deuterium Exchange-massespektrometri til at studere proteiners struktur og Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Et modificeret gær-et hybrid system til studier af heteromere proteinkompleks-DNA-interaktioner

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Biotinylated celle-gennemtrængende peptider til at studere intracellulær Protein-protein interaktioner

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Et fluorescens udsving spektroskopi Assay af Protein-Protein interaktioner på celle-celle kontakter

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Et Fluorogenic peptid kavalergang Assay til at screene for proteolytiske aktivitet: ansøgninger om coronavirus spike protein aktivering

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Anvendelse af Biolayer interferometri (BLI) til undersøgelse af protein-protein interaktioner i transkriptionen

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Anvendelse af mikroskala termoforese til måling af protein-lipid interaktioner

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

I Vivo Hydroxyl Radical Protein Footprinting for studiet af proteininteraktioner i Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...