1、I.IntroductionII.Yeast Molecular TechniquesIII.Constructing a Reporter Vector for One-Hybrid AnalysisIV.Generating a cDNA LibraryV.Constructing&Screening a One-Hybrid LibraryVI.Analyzing Positive InteractionsYeast one hybrid systemI.Introduction oOne-Hybrid System provides an in vivo genetic assay u
2、sed for isolating novel genes encoding proteins that bind to a target,cis-acting regulatory elementoProtein-DNA interactionI.INTRODUCTIONThe differences between yeast one&two hybridII.Yeast Molecular TechniquesYeast strainsA.GenotypesB.PhenotypesII.Yeast Molecular TechniquesYeast strain：Y187（MAT）Can
3、t synthesis His、Leu、Trp by itself。YeastIncubateYeast selection medium-His or-Leu or-TrpYeast cant growth on selection mediumII.Yeast Molecular TechniquesYeast strainsC.Mating type compatibilitiesoY187(MAT)can mate with AH109,HF7c,CG-1945,Y190,or SFY526(all MATa).D.Reporter genesGAL1 UASGAL TATALac Z
4、II.Yeast Molecular TechniquesYeast strainsE.Leaky HIS3 expressiono3-AT:3-amino-1,2,4-triazole is a competitive inhibitor of the yeast HIS3 protein(His3p).3-AT is used to inhibit low levels of His3p expressed in a leaky manner and thus to suppress background growth on SD medium lacking histidine.oSom
5、e yeast strains have relatively high basal levels of His3p.II.Yeast Molecular TechniquesMain difference between yeast and E.coli cultureYeastE.Coli46 days16 hoursYeastE.Coli3037YeastE.ColiYPDLBoIncubation timeoIncubation temperatureoCulture mediumPick individual colonies.To 2%glucosesterile(40%stock
6、 solution)coli cultureIntroductionSD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+120mM 3-AT10 l 10X BD Advantage 2 PCR Buffer0mM 20mM 40mM 60mMHeat shock for 15 min in a 42C water bath.Constructing&Screening One-Hybrid LibrariesBackground HIS3 Expression of ICEr2Yeast strainsGenerating a cDNA Librar
7、yGood Laboratory Practices1 mg of carrier DNA.How BD SMART cDNA Synthesis and Amplification WorksSD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+120mM 3-ATReporter genesFigure 3.SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+120mM 3-ATConstructing a Reporter Vector for One-Hybrid AnalysisTo optimize
8、the 3-AT concentration in your selection medium:Use the lowest concentration of 3-AT that,after one week,allows only small(1 mm)colonies to grow.Too much 3-AT in the medium can kill freshly transformed cells.YEAST MEDIAA.YPD(YPDA)mediumo20 g/L Difco peptoneo10 g/L Yeast extracto20 g/L Agar(for plate
9、s only)o(To 0.003%0.2%adenine hemisulfate Solution)opH to 6.5 oTo 2%glucosesterile(40%stock solution)II.Yeast Molecular TechniquesYEAST MEDIA B.SD medium(1L)o6.7 g Yeast nitrogen base without amino acidso20 g Agar(for plates only)o850 ml H2OopH to 5.8 o100 ml of the appropriate sterile 10X Dropout S
10、olutiono3-AT(1 M 3-AT stock solution)oTo 2%glucosesterile(40%stock solution)II.Yeast Molecular TechniquesC.10X Dropout(DO)Solution Nutrient 10X Concentration L-Adenine hemisulfate salt 200 mg/L L-Arginine HCl 200 mg/L L-Histidine HCl monohydrate 200 mg/L L-Isoleucine 300 mg/L L-Leucine 1000 mg/L L-L
11、ysine HCl 300 mg/L L-Methionine 200 mg/L L-Phenylalanine 500 mg/L L-Threonine 2000 mg/L L-Tryptophan 200 mg/L L-Tyrosine 300 mg/L L-Uracil 200 mg/L L-Valine 1500 mg/LAutoclaveII.Yeast Molecular TechniquesYEAST MEDIASuppliedo10 g Leu DO Supplement(to measure the transformation efficiency of the libra
12、ry plasmid)o10 g Trp DO Supplement(to measure the transformation efficiency of the reporter plasmid)o10 g Leu/Trp DO Supplement(to measure the number of clones screened)o10 g His/Leu/Trp DO Supplement(select for one-hybrid interactions）UnsuppliedoHis/Trp DO Supplement（to measure a pHIS2 reporter pla
13、smid for background growth）II.Yeast Molecular TechniquesPreparation of Competent Yeast CellsLiAc Method1.Inoculate one colony(4 weeks old,23 mm in diameter)into 3 ml of YPDA medium in a sterile,15-ml centrifuge tube.2.Incubate at 30C with shaking for 8 hr.3.Transfer 5 l of the culture to a 250-ml fl
14、ask containing 50 ml of YPDA.4.Incubate at 30C with shaking at 230250 rpm for 1620 hr.The OD600 should reach 0.150.3.5.Centrifuge the cells at 700 x g for 5 min at room temperature.6.Discard the supernatant and resuspend the cell pellet in 100 ml of YPDA.7.Incubate at 30C for 35 hr(OD600=0.40.5).8.C
15、entrifuge the cells at 700 x g for 5 min at room temperature.9.Discard the supernatant and resuspend the cell pellet in 60 ml of sterile,deionized H2O.10.Centrifuge the cells at 700 x g for 5 min at room temperature.11.Discard the supernatant and resuspend the cells in 3 ml of 1.1X TE/LiAc Solution.
16、12.Split the resuspension between two 1.5-ml microcentrifuge tubes(1.5 ml per tube).13.Centrifuge each tube at high speed for 15 sec.14.Discard the supernatant and resuspend each pellet in 600 l of 1.1X TE/LiAc Solution.oNote:1.Competent cells should be used for transformation immediately following
17、preparation;however,if necessary they can be stored at room temperature for a few hours without significantly affecting the competency.o2.on iceSmall-scale LiAc Yeast Transformation Procedure12.Add 0.1 mg of plasmid DNA and 0.1 mg of herring testes carrier DNA to a fresh 1.5-ml tube and mix.13.Add 0
18、.1 ml of yeast competent cells to each tube and mix well by vortexing.14.Add 0.6 ml of sterile PEG/LiAc solution to each tube and vortex at high speed for 10 sec to mix.15.Incubate at 30C for 30 min with shaking at 200 rpm.16.Add 70 ml of DMSO.Mix well by gentle inversion.Do not vortex.17.Heat shock
19、 for 15 min in a 42C water bath.18.Chill cells on ice for 12 min.19.Centrifuge cells for 5 sec at 14,000 rpm at room temperature.Remove the supernatant.20.Resuspend cells in 0.5 ml of sterile 1X TE buffer.21.Plate 100 ml on each SD agar plate that will select for the desired transformants.To ensure
20、thatyou will obtain a plate with well-separated colonies,also spread 100 ml of a 1:1000,1:100,and1:10 dilution on 100-mm SD agar plates.These will also provide controls for(co)transformation efficiency.22.Incubate plates,up-side-down,at 30C until colonies appear.Notes:oFor simultaneous cotransformat
21、ion,use 0.1 mg of each plasmid,in addition to the 0.1 mg of carrier DNA.ocarrier DNA.oV1/5oGlassoAiro30II.Yeast Molecular TechniquesTroubleshooting of Yeast TransformationoTransformation efficiency:104 cfu/mg(single type of plasmid);103cfu/mg(two types of plasmids)1.Suboptimal plasmid preparationoUs
22、ing more(up to 0.5 mg)of the plasmid DNAoCheck the purity of the DNA and,if necessary,repurify it by ethanol2.Suboptimal carrier DNAoIf transformation efficiencies are declining in successive experiments,the carrier DNA may be renaturing.Reboil the carrier DNA for 20 min,and then chill it quickly in
23、 an ice-water bath.Troubleshooting of Yeast Transformation3.Suboptimal yeast competent cellsoInoculate with a fresh colonyoCheck the liquid medium to make sure it was made correctlyoThe addition of adenine hemisulfate to YPD(in Steps E.3 and E.5)will enhance the growth of yeast strains that contain
24、the ade2-101 mutation.oCheck the concentration of the resuspended competent cells.If the cell concentration is 1 x 109/ml,spin the cells down again(at 1,000 x g for 5 min)and resuspend them in a smaller volume of 1X TE/LiAc bufferoPrepare all reagents using sterile,deionized,distilled wateroSD plate
25、:2-4d at RT or 3h at 30III.Constructing a Reporter Vector for One-Hybrid AnalysisOne-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target,cis-acting regulatory elementLeaky HIS3 expression100 l Total volumeSD/-Leu SD/-Trp SD/-Leu/-Trp
26、 SD/-Leu/-Trp/-His+120mM 3-ATTroubleshooting of Yeast TransformationIncubation timeRescue the Library cDNA InsertRetest the Interaction In Vitro1 mg of plasmid DNA and 0.Analyzing Positive InteractionsConstructing a Reporter Vector for One-Hybrid AnalysisPhenotypesPCR Fragment Purification using aga
27、rose gel electrophoresis.To 2%glucosesterile(40%stock solution)If the PCR product consists of more than one band,see Part D,below.Compare the sequence with that of other proteins in GenBank,EMBL,or other databases.Background0 l(2 units)RNase H.Yeast strainsPCR Fragment Purification using agarose gel
28、 electrophoresis.技技 术术 路路 线线A.BackgroundnIdentified a true or putative target elementnA construct composed of one or more tandem copies of your targetnConstructs should be tested for background(leaky)HIS3 expression before you start a one-hybrid analysisIII.Constructing a Reporter Vector for One-Hyb
29、rid AnalysisB.Synthesize Your Target ElementThe reporter should contain at least three tandem copies of the DNA targetThe most convenient and reliable method for generating them to be oligonucleotide synthesisIII.Constructing a Reporter Vector for One-Hybrid AnalysisC.Insert Your DNA Target into the
30、 Multiple Cloning Site of pHIS2 退火，连接，转化，酶切鉴定，测序D.Test your Target-Reporter Construct for Background HIS3 ExpressionIII.Constructing a Reporter Vector for One-Hybrid AnalysisBackground HIS3 Expression of ICEr2 0mM 20mM 40mM 60mM 80mM 100mM 110mM 120mMIV.Generating a cDNA LibraryA.How BD SMART cDNA S
31、ynthesis and Amplification WorksIV.Generating a cDNA LibraryB.Good Laboratory PracticesoWhen resuspending pellets or mixing reactions,gently pipet the solution up and down or tap the bottom of the tube.Spin briefly to bring contents to the bottom of the tube.Do not vortex samples when resuspending p
32、ellets;vortexing may shear your cDNA.oPerform all reactions on ice,unless otherwise indicated.oDo not increase the size(volume)of any of the reactions.All components have been optimized for the volumes specified.oIn preparing your reactions,use the Deionized H2O supplied.IV.Generating a cDNA Library
33、C.RNA IsolationoThe minimum amount of starting material for cDNA synthesis is 100 ng of total RNA or 25 ng of poly A+RNA.Use the higher starting amounts of RNA shown in the table.D.RNA AnalysisIV.Generating a cDNA Library图图5 拟南芥拟南芥叶片总叶片总RNA的提取的提取Figure.5 Isolation of total RNA from Arabidopsis18S28S
34、E.Synthesize First-Strand cDNA using an Oligo(dT)Primer1.Combine the following reagents in a sterile 0.25-ml microcentrifuge tube:12 l RNA sample(0.0251.0 g poly A+or 0.102.0 g total RNA),1.0 l CDS III Primer,12 l Deionized H2O to bring volume up to 4.0 l.,4.0 l Total volume2.Mix contents and spin b
35、riefly.3.Incubate at 72C for 2 min.4.Cool on ice for 2 min.5.Spin briefly.6.Add the following to the reaction tube:2.0 l 5X First-Strand Buffer,1.0 l DTT(20 mM),1.0 l dNTP Mix(10 mM),1.0 l MMLV Reverse Transcriptase,9.0 l Total volume7.Mix gently by tapping.Spin briefly.8.Incubate at 42C for 10 min.
36、9.Add 1.0 l BD SMART III Oligonucleotide.10.Incubate at 42C for 1 hr in an air incubator or hot-lid thermal cycler.11.Place the tube at 75C for 10 min to terminate first-strand synthesis.12.Cool the tube to room temperature,then add 1.0 l(2 units)RNase H.13.Incubate at 37C for 20 min.14.If you plan
37、to proceed directly to the LD-PCR step,take a 2-l aliquot from the first-strand synthesis and place it in a clean,prechilled,0.5-ml tube.Place the tube on ice,and proceed to Section H.If you used mineral oil in your first-strand reaction tube,be sure to take the 2-l sample from the bottom of the tub
38、e to avoid the oil.15.Any first-strand reaction mixture that is not used right away should be placed at 20C.F.Amplify ds cDNA by Long Distance PCR(LD-PCR)2 l First-Strand cDNA70 l Deionized H2O10 l 10X BD Advantage 2 PCR Buffer2 l 50X dNTP Mix2 l 5 PCR Primer2 l 3 PCR Primer10 l 10X GC-Melt Solution
39、2 l 50X BD Advantage 2 Polymerase Mix100 l Total volume 95C 30 sec x cyclesa:95C 10 sec68C 6 min*68C 5 minLD-PCR反应条件LD-PCR反应体系G.Purify ds cDNA with a BD CHROMA SPIN TE-400 ColumnV.Constructing&Screening a One-Hybrid LibraryA.Cotransform Yeast Strain Y187 with ds cDNA,pGADT7-Rec2,and pHIS2/target DNA
40、.oNote:The combined volume of these DNA components should not exceed 60 l,or 1/10 the volume of the competent cells.B.Select for One-Hybrid InteractionsoSpread 100 l of a 1:10,1:100,and 1:1,000 dilution onto 100-mm SD/Leu,SD/Trp,and SD/Leu/Trp agar plates.Incubate at 30C for 37 days until colonies a
41、ppear.oRestreak the His+colonies on fresh SD/His/Leu/Trp+optimal 3-AT.V.Constructing&Screening One-Hybrid LibrariesIdentified a true or putative target elementAnalyzing Positive Interactions0 l MMLV Reverse Transcriptase,9.In preparing your reactions,use the Deionized H2O supplied.Yeast strainsAnaly
42、zing Positive Interactions10 g Leu/Trp DO Supplement(to measure the number of clones screened)One-Hybrid Controls7 g Yeast nitrogen base without amino acidsYeast Molecular TechniquesIncubate plates,up-side-down,at 30C until colonies appear.Restreak the His+colonies on fresh SD/His/Leu/Trp+optimal 3-
43、AT.Incubation temperatureMutant(AAAAAA)INTRODUCTIONThe differences between yeast one&two hybridGood Laboratory Practices100 l Total volumeCompare the sequence with that of other proteins in GenBank,EMBL,or other databases.Incubation temperatureC.One-Hybrid ControlsV.Constructing&Screening One-Hybrid
44、 Libraries负对照正对照 SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+10mM 3-AT SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+10mM 3-ATOne-Hybrid ControlsOne-Hybrid InteractionsICEr2 SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+120mM 3-ATVI.Analyzing Positive InteractionsVI.Analyzing Positive Interacti
45、onsA.Retest the PhenotypeoRestreak the positive colonies on SD dropout plates 23 times to segregate the AD/library plasmids.oReplica plate or transfer well-isolated colonies to SD/His/Leu/Trp plates containing different concentrations of 3-AT to verify that they maintain the correct phenotype and to
46、 test the strength of the interaction.VI.Analyzing Positive InteractionsB.Rescue the Library cDNA InsertoPlasmid IsolationC.Analyze the cDNA Insert by Agarose/EtBr Gel ElectrophoresisoIf the PCR product consists of more than one band,see Part D,below.oIf the PCR product consists of a single band:nPr
47、epare a new master plate with a representative clone from each group.nIf you are satisfied with the number of unique clones,prepare a glycerol stock of each unique type.Store at 80C.nPurify the PCR product using any suitable method.nSequence the cDNA InsertVI.Analyzing Positive InteractionsD.Segrega
48、tion of AD/Library Plasmids Segregation in Yeast:oRestreak positive colonies on SD dropout plates 23 times to segregate the AD/library plasmidsoReplica plate or transfer well-isolated colonies to the appropriate SD dropout plates to verify that they maintain the correct phenotype.Segregation in Bact
49、eria:oIsolate the plasmids from yeastoTransform E.coli DH5 cells with the plasmid preparation and select on LB/amp plates.Pick individual colonies.oPCR Fragment Purification using agarose gel electrophoresis.oCompare the sequence with that of other proteins in GenBank,EMBL,or other databases.VI.Anal
50、yzing Positive InteractionsE.Retest the Interaction In Vitrooelectrophoretic mobility-shift assay(EMSA)nTranscribe and translate the HA epitope-tagged fusion protein in vitro using the T7 promoter in the AD vector pGADT7-Rec2.nPerform an EMSA assay with your wild-type targets.VI.Analyzing Positive I
