酵母单杂交系统及其实验操作.ppt

酵母单杂交系统及其实验操作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 system酵母单杂交系统及其实验操作I. Introduction酵母单杂交系统及其实验操作 oOne-Hybrid System provides an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory elementoProtein-DNA interaction酵母单杂交系统及其实验操作The differences between yeast one & two hybrid酵母单杂交系统及其实验操作酵母单杂交系统及其实验操作II. Yeast Molecular Techniques酵母单杂交系统及其实验操作Yeast strainsA. GenotypesB. PhenotypesII.Yeast Molecular Techniques酵母单杂交系统及其实验操作Yeast strain：Y187（MATα）Can’t synthesis His、 Leu、Trp by itself。

YeastIncubateYeast selection medium-His or -Leu or -TrpYeast can’t growth on selection mediumII.Yeast Molecular Techniques酵母单杂交系统及其实验操作Yeast strainsC. Mating type compatibilitiesoY187 (MATα) can mate with AH109, HF7c, CG-1945, Y190, or SFY526 (all MATa).D. Reporter genesGAL1 UASGAL TATALac ZII.Yeast Molecular Techniques酵母单杂交系统及其实验操作Yeast 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.oSome yeast strains have relatively high basal levels of His3p.II.Yeast Molecular Techniques酵母单杂交系统及其实验操作Main difference between yeast and E.coli cultureYeastE.Coli4~6 days16 hoursYeastE.Coli30℃37℃YeastE.ColiYPDLBoIncubation timeoIncubation temperatureoCulture medium酵母单杂交系统及其实验操作To optimize 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.酵母单杂交系统及其实验操作YEAST MEDIAA. YPD(YPDA) mediumo20 g/L Difco peptoneo10 g/L Yeast extracto20 g/L Agar (for plates only)o(To 0.003% 0.2% adenine hemisulfate Solution)opH to 6.5 oTo 2% glucosesterile (40% stock solution)II.Yeast Molecular Techniques酵母单杂交系统及其实验操作YEAST 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 Solutiono3-AT(1 M 3-AT stock solution)oTo 2% glucosesterile (40% stock solution)II.Yeast Molecular Techniques酵母单杂交系统及其实验操作C.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-Lysine 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 MEDIA酵母单杂交系统及其实验操作Suppliedo10 g –Leu DO Supplement(to measure the transformation efficiency of the library 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 ）Unsuppliedo–His/–Trp DO Supplement（to measure a pHIS2 reporter plasmid for background growth）II.Yeast Molecular Techniques酵母单杂交系统及其实验操作Preparation of Competent Yeast Cells—LiAc Method1. Inoculate one colony (< 4 weeks old, 2–3 mm in diameter) into 3 ml of YPDA medium in a sterile,15-ml centrifuge tube.2. Incubate at 30°C with shaking for 8 hr.3. Transfer 5 μl of the culture to a 250-ml flask containing 50 ml of YPDA.4. Incubate at 30°C with shaking at 230–250 rpm for 16–20 hr. The OD600 should reach 0.15–0.3.5. 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 30°C for 3–5 hr (OD600 = 0.4–0.5).8. Centrifuge 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.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 μl of 1.1X TE/LiAc Solution.oNote: 1.Competent cells should be used for transformation immediately following preparation; however, if necessary they can be stored at room temperature for a few hours without significantly affecting the competency.o2. on ice酵母单杂交系统及其实验操作Small-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 0.1 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 30°C 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 for 15 min in a 42°C water bath.18. Chill cells on ice for 1–2 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 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 30°C until colonies appear.酵母单杂交系统及其实验操作Notes:oFor simultaneous cotransformation , use 0.1 mg of each plasmid , in addition to the 0.1 mg of carrier DNA.ocarrier DNA.oV<1/5oGlassoAiro30℃II.Yeast Molecular Techniques酵母单杂交系统及其实验操作Troubleshooting of Yeast TransformationoTransformation efficiency: 104 cfu/mg(single type of plasmid); 103cfu/mg(two types of plasmids)1. Suboptimal plasmid preparationoUsing 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 an ice-water bath.酵母单杂交系统及其实验操作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 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 : 2-4d at RT or 3h at 30℃酵母单杂交系统及其实验操作III.Constructing a Reporter Vector for One-Hybrid Analysis酵母单杂交系统及其实验操作技技 术术 路路 线线酵母单杂交系统及其实验操作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-Hybrid Analysis酵母单杂交系统及其实验操作B. 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 Analysis酵母单杂交系统及其实验操作C. Insert Your DNA Target into the 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 Analysis酵母单杂交系统及其实验操作Background HIS3 Expression of ICEr2 0mM 20mM 40mM 60mM 80mM 100mM 110mM 120mM酵母单杂交系统及其实验操作IV. Generating a cDNA Library酵母单杂交系统及其实验操作A. How BD SMART cDNA Synthesis and Amplification WorksIV.Generating a cDNA Library酵母单杂交系统及其实验操作B. 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 pellets; 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酵母单杂交系统及其实验操作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酵母单杂交系统及其实验操作E. Synthesize First-Strand cDNA using an Oligo (dT) Primer1. Combine the following reagents in a sterile 0.25-ml microcentrifuge tube:1–2 μl RNA sample (0.025–1.0 μg poly A+ or 0.10–2.0 μg total RNA),1.0 μl CDS III Primer,1–2 μl Deionized H2O to bring volume up to 4.0 μl.,4.0 μl Total volume2. Mix contents and spin briefly.3. Incubate at 72°C for 2 min.4. Cool on ice for 2 min.5. Spin briefly.6. Add the following to the reaction tube:2.0 μl 5X First-Strand Buffer,1.0 μl DTT (20 mM),1.0 μl dNTP Mix (10 mM ),1.0 μl MMLV Reverse Transcriptase,9.0 μl Total volume7. Mix gently by tapping. Spin briefly.8. Incubate at 42°C for 10 min.9. Add 1.0 μl BD SMART III Oligonucleotide.10. Incubate at 42°C for 1 hr in an air incubator or hot-lid thermal cycler.11. Place the tube at 75°C for 10 min to terminate first-strand synthesis.12. Cool the tube to room temperature, then add 1.0 μl (2 units) RNase H.13. Incubate at 37°C for 20 min.14. If you plan to proceed directly to the LD-PCR step, take a 2-μl 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-μl sample from the bottom of the tube to avoid the oil.15. Any first-strand reaction mixture that is not used right away should be placed at –20°C. 酵母单杂交系统及其实验操作F. Amplify ds cDNA by Long Distance PCR (LD-PCR)2 μl First-Strand cDNA70 μl Deionized H2O10 μl 10X BD Advantage 2 PCR Buffer2 μl 50X dNTP Mix2 μl 5' PCR Primer2 μl 3' PCR Primer10 μl 10X GC-Melt Solution2 μl 50X BD Advantage 2 Polymerase Mix100 μl Total volume• 95°C 30 sec• x cyclesa:95°C 10 sec68°C 6 min*• 68°C 5 minLD-PCR反应条件LD-PCR反应体系酵母单杂交系统及其实验操作G. Purify ds cDNA with a BD CHROMA SPIN™ TE-400 Column酵母单杂交系统及其实验操作V. Constructing & Screening a One-Hybrid Library酵母单杂交系统及其实验操作A. Cotransform Yeast Strain Y187 with ds cDNA, pGADT7-Rec2, and pHIS2/target DNA.oNote: The combined volume of these DNA components should not exceed 60 μl, or 1/10 the volume of the competent cells.B. Select for One-Hybrid InteractionsoSpread 100 μl 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 30°C for 3–7 days until colonies appear.oRestreak the His+ colonies on fresh SD/–His/–Leu/–Trp + optimal [3-AT].V.Constructing & Screening One-Hybrid Libraries酵母单杂交系统及其实验操作C. One-Hybrid ControlsV.Constructing & Screening One-Hybrid Libraries酵母单杂交系统及其实验操作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 Controls酵母单杂交系统及其实验操作One-Hybrid InteractionsICEr2 SD/-Leu SD/-Trp SD/-Leu/-Trp SD/-Leu/-Trp/-His+120mM 3-AT酵母单杂交系统及其实验操作VI. Analyzing Positive Interactions酵母单杂交系统及其实验操作VI.Analyzing Positive Interactions酵母单杂交系统及其实验操作A. Retest the PhenotypeoRestreak the positive colonies on SD dropout plates 2–3 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 test the strength of the interaction.VI.Analyzing Positive Interactions酵母单杂交系统及其实验操作B. 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:nPrepare 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 –80°C.nPurify the PCR product using any suitable method. nSequence the cDNA InsertVI.Analyzing Positive Interactions酵母单杂交系统及其实验操作D. Segregation of AD/Library Plasmids Segregation in Yeast:oRestreak positive colonies on SD dropout plates 2–3 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 Bacteria:oIsolate the plasmids from yeastoTransform E. coli DH5α 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.Analyzing Positive Interactions酵母单杂交系统及其实验操作E. 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 Interactions酵母单杂交系统及其实验操作。