DupLEX-A YEAST TWO
Contents
1. 10 70 ethanol Mix 350 ml of absolute ethanol with 150 ml of distilled water and store at 20 C 11 10 glycerol Mix 100 ml of glycerol with 900 ml of distilled water and autoclave for 20 minutes 12 50 glycerol Mix 250 ml of glycerol with 250 ml of distilled water and autoclave for 20 minutes IV Library Information All DupLEX A Yeast Two Hybrid System libraries are made in the B42 activation domain HA tag expression vector pJG4 5 The cDNA is made by oligo d T priming and is cloned unidirectionally between the EcoRI and XhoI sites of pJG4 5 see Appendix for details The libraries are provided as ready to use plasmid DNA and also as plasmid containing bacterial cells 13 V Culturing of Yeast The budding yeast Saccharomyces cerevisiae is very amenable to genetic and molecular biological methodologies due to its ability to be transformed by foreign DNA its highly efficient system of homologous recombination and its relatively rapid rate of growth Whereas E coli has a generation time of 30 45 minutes most yeast strains can double in 90 120 minutes As with E coli yeast can be grown on plates or in liquid culture However antibiotics which work on E coli do not work on yeast making good sterile technique mandatory when working with yeast Finally the optimum growth temperature for yeast is 28 32 C VI DupLEX A Yeast Two Hybrid System Protocol A Making the LexA bait protein expression vector 1 U2. Ap inducible GAL1 promoter expresses B42 HA tag and is followed by a polylinker for making target fusion protein expression libraries from cDNA 10 ug Control plasmids 2 ug each pRHFM1 HIS3 2 um Ap ADH promoter expresses Lex A homeodomain of bicoid fusion used as a positive control in the repression assay and a negative control in the DupLEX A screen pSH17 4 HIS3 2 um Ap ADH promoter expresses LexA GAL4 activation domain used as a positive control in the DupLEX A screen pEG202 Max expresses LexA Max fusion constitutively used as a negative control when testing isolated target proteins or as a positive control in the repression assay pBait constitutively expresses a LexA bait fusion protein that interacts with the fusion protein from pTarget see below can also be used as a negative control when testing isolated target proteins or as a positive control in the repression assay pTarget expresses galactose dependently a B42 target fusion protein that interacts with the fusion protein from pBait see above Primers 10 ug each Note The concentration of each oligo is approximately 80 uM 5 bait fusion primer 5 CGT CAG CAG AGC TTC ACC 3 used to determine the sequence of the junction between LexA and the bait 5 target fusion primer 5 CTG AGT GGA GAT GCC TCC 3 used to determine the reading frame and identity of positive clones also can be used with 3 target primer to amplify clone
3. in 100 ml of distilled water Mix the two solutions add 0 8 ml of 100 mg ml X gal in N N dimethyl formamide and pour plates a LB medium Dissolve 20 g of dry LB Broth Lennox from Fisher in 1 liter of distilled water and autoclave for 15 minutes For plates add 15 g of agar per liter of medium before autoclaving and cool to 50 C before pouring b LBA medium ampicillin selection 10 Cool the LB medium above to 50 C and add 2 ml of 50 mg ml ampicillin in distilled water filter sterilized per liter of medium Mix c LBK medium kanamycin selection Cool the LB medium above to 50 C and add 5 ml of 10 mg ml kanamycin sulfate in distilled water filter sterilized per liter of medium Mix d Minimal trp medium B SOLUTIONS 1 10 X TE Prepare the following stocks autoclaved i 20 magnesium sulfate ii 4 mg ml uracil iii 4 mg ml histidine iv 4 mg ml leucine v 20 glucose Prepare the following filter sterilized solutions vi 50 mg ml kanamycin sulfate vii 1 thiamine hydrochloride Autoclave the following two solutions separately viii 15 g agar in 800 ml distilled water ix 10 5 g potassium phosphate dibasic 4 5 g potassium phosphate monobasic 1 g ammonium sulfate 0 5 g sodium citrate 160 ml distilled water Mixing Cool solutions viii and ix to 50 C mix and quickly add 1 ml of solution i 10 ml of solution ii 10 ml of solution iii 10 ml of solution iv 10 m
4. Mann 835 269 3 For yeast transformations item vendor catalog lithium acetate Fisher AC19984 2500 polyethylene glycol 3350 Sigma P146 3 dimethyl sulfoxide Fisher D136 1 hydrochloric acid Fisher A144 500 tris base Fisher BP152 5 EDTA Fisher 02793 500 4 For bacterial transformations item vendor catalog glycerol Sigma G 5516 ElectroMax DH10B Gibco BRL 18290 015 competent cells SOC medium Gibco BRL 15544 018 electroporator Gibco BRL 11613 015 5 For rescuing plasmids from yeast item vendor catalog glass beads acid washed Sigma G8772 triton X 100 Fisher BP151 100 sodium acetate Fisher S209 500 ethanol Aldrich 18 738 0 phenol Fisher BP17501 400 chloroform Fisher BP1145 1 isoamyl alcohol Fisher BP1150 500 SDS Sigma L 4509 6 Filter assay for B galactosidase and yeast X gal plates item vendor catalog sodium phosphate Fisher BP329 500 monobasic sodium phosphate Mallinckrodt 7914 dibasic potassium chloride Mallinckrodt 6858 B mercaptoethanol Fisher 03446I 100 nylon membrane filters MSI NO4SP09025 X gal Gold BioTech X4281C N N dimethyl formamide Fisher BP1160 500 III Recipes for media and solutions A MEDIA 1 Yeast a YPD rich medium 20 g peptone 10 g yeast extract 20 g glucose one pellet 0 1 g NaOH if for plates 20 g agar if for plates Add I liter of distilled water and autoclave for 20 minutes For plates cool to 50 C before pouring b YNB ura his leu trp s
5. by PCR 3 target fusion primer 5 GCC GAC AAC CTT GAT TG 3 used to determine the identity of positive clones also can be used with 5 target primer to amplify clone by PCR Other pJG4 6 TRP1 2 um Ap similar to pJG4 5 but without B42 activation domain used to express an isolated target protein in yeast 10 ug sonicated salmon sperm DNA 5 mg ml prepared specially for yeast transformation 10 mg total CARRIER DNA E coli strain KC8 DupLEX A System Manual B ADDITIONAL MATERIALS REQUIRED not supplied Note The specific materials listed below are the ones we have tested in the DupLEX A system Similar items from other sources may be interchangeable 1 Yeast growth media vendor catalog peptone Difco 0118 17 0 agar Difco 0145 17 0 yeast extract Difco 0127 17 9 yeast nitrogen base Difco 0919 15 3 w o amino acids raffinose Sigma R 0250 dextrose glucose Fisher D16 1 galactose Sigma G 0750 glucose free dropout mix BIO101 4540 022 his ura trp leu uracil Sigma U 0750 leucine Sigma L 5652 tryptophan Sigma T 0254 histidine Fisher BP382 100 2 Bacterial growth media vendor catalog LB broth Difco 0402 07 0 magnesium sulfate Fisher BP213 1 potassium phosphate Mallinckrodt 7100 monobasic potassium phosphate Fisher BP363 500 dibasic sodium citrate Fisher BP327 1 thiamine hydrochloride Fisher BP892 100 ammonium sulfate Fisher BP212R 1 kanamycin Fisher BP906 5 ampicillin Boehringer
6. original bait you used to make sure that it still interacts Once you have a positive that passes all the specificity tests you should transform the DNA into a different E coli strain do a DNA prep and sequence a portion of your clone You can then do a database search to see if you can identify your protein It is imperative to realize that even a clone that passes all the tests could still be a false positive For example in some instances clones have been obtained that appear to interact specifically with a certain bait but it was already known that the two molecules are located in different parts of the cell therefore they are not real interactors That is why the yeast two hybrid system should be considered a relatively quick and easy method of obtaining the cloned gene for a protein which may interact with your protein of interest Once you have the clone you still need to do more to show that it is biologically relevant Note that pJG4 6 is included in the kit it is similar to pJG4 5 except that it does not contain LexA It can be used to express your target protein alone in yeast HAPPY FISHING VII APPENDIX 1 pEG202 polylinker sequence unique sites shown 5 CTG GAA TTC CCG GGG ATC CGT CGA CCA TGG CGG CCG CTC LexA EcoRI BamHI Ncol Notl 24 GAG TCG ACC TGC AGC 3 2 pJG4 5 polylinker sequence 5 CCC GAA TTC GGC CGA CTC GAG AAG 3 EcoRI Xhol 3 pNLexA polylinker sequence 5 G AAT TCG CGG CCG CC
7. DupLEX A YEAST TWO HYBRID SYSTEM User s Manual Version 1 2 1 97 OriGene Technologies Inc 13 Taft Court Suite 111 Rockville MD 20850 1 888 267 4436 FAX 301 340 9254 DupLEX A Yeast Two Hybrid System Index I Introduction II List of components additional materials required III Recipes for media and solutions IV Library information V Checking and storing yeast strains VI Screening libraries VII Appendix I Introduction The DupLEX A system is a LexA based version of the yeast two hybrid system originally developed by Fields and Song The yeast two hybrid system has proven to be a powerful tool for identifying proteins from an expression library which can interact with one s protein of interest The DupLEX A system was developed as a more versatile and more accurate version of the yeast two hybrid system The two hybrid system of Fields and Song exploits the fact that a yeast transcriptional activator protein Gal4p has a separable DNA binding domain and activation domain neither domain can activate transcription on its own Transcriptional activation is detected only when the binding domain is bound to its DNA recognition sequence and is also tethered to the activation domain The two hybrid system involves fusing the Gal4p binding domain with a protein X and the Gal4p activation domain with a protein Y If X and Y interact then a functional Gal4p is restored and transcripti
8. Shake at 30 C for 4 hours to induce the GAL1 promoter b Do serial dilutions of the transformants in YNB gal his ura trp and plate 100 ul of each dilution onto separate YNB gal his ura trp plates Incubate at 30 C for 2 3 days until colonies are visible c Calculate the number of colony forming units cfu per frozen aliquot of yeast transformants 2 Screen for Leu colonies a To fully screen all the transformants you should screen about 5 7 times the number of original transformants obtained see section VLE 11 Therefore if you obtained 3 x 10 transformants originally you would want to plate about 2 x 10 cfu from step 1 above now Thaw enough of the frozen transformants equal to that number of cfu b The number of cfu per 1 ml frozen aliquot can be used to estimate an OD of the aliquot since 1 OD 2 x 10 yeast cells That is ft of cfu 2 x 10 OD of the aliquot Dilute the appropriate amount of frozen transformants with YNB gal his ura trp leu medium down to approximately 1 x 10 cells ml OD 0 5 Shake at 30 C for 4 hours c Plate 100 ul aliquots onto 100 mm YNB gal his ura trp leu plates You will be screening 1 x 10 cfu per plate do not exceed this number Incubate at 30 C for 2 days until colonies appear 20 d Pick colonies onto a YNB gal his ura trp leu master plate and incubate at 30 C for a few days until colonies appear Go back to the plates from step c above and d
9. T CGA GGG ATC CAA TTC ATG AAA GCG 3 4 pEG202 NLS polylinker sequence 5 GTG GAA TTC CCG GGG ATC CGT CGA CCT GCA GCC 3 NOTE FOR RESEARCH PURPOSES ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USAGE 25 26
10. bait plasmid and pSH18 34 into 0 5 ml of sterile distilled water Vortex Dilute 100 ul into I ml of sterile distilled water Vortex This is Dilution 1 Do three more serial 1 10 dilutions Dilutions 2 4 such that if Dilution 1 is considered undiluted Dilution 2 1 10 diluted Dilution 3 1 100 diluted and Dilution 4 1 1000 diluted b Plate 100 ul of each of Dilutions 1 4 onto YNB gal his ura plates and onto YNB gal his ura leu plates Incubate at 300C for 16 1 2 days You should see colonies on the his ura plates but not on the his ura leu plates Note galactose plates are used in this experiment since that is the carbon source that will be used during the LEU2 selection step of the large scale screen If you do obtain many colonies on the his ura leu plates then your bait is autoactivating and you should re do the assays using your bait in strains EGY 194 and EGY 188 EGY40 is included as a negative control strain In addition three different sensitivity lacZ reporter plasmids are included pSH18 34 gt pJK103 gt pRB1840 most sensitive gt least sensitive Once you are convinced that your bait fusion can enter the nucleus and bind to LexA operators without autoactivating either of the two reporter genes then you are ready to perform a large scale library screen Note that for an unknown reason some baits can autoactivate the reporter genes in a large scale screen even when they did not autoactivate in small scale t
11. e freeze at 70 C Take out thaw and place yeast side up on Whatman filter paper pre cut above Incubate at 30 C for 2 hours Expected results The colonies containing pSH17 4 should turn blue the colonies containing pRFHM1 should not turn blue and the colonies containing the pEG202 Bait plasmid may or may not turn blue If they do not turn blue then the Bait does not autoactivate reporter gene expression and can be used for screening in the yeast strain EGY48 If the clones containing pEG202 Bait do turn blue in the above assay then the test should be repeated using pJK103 or pRB1840 instead of pSH18 34 If the Bait does not autoactivate in one of these strains use that strain to perform the screen If the Bait still autoactivates even in the least sensitive strain then you must subclone only portions of the gene encoding your protein into pEG202 and test for a portion that does not autoactivate C Testing the autoactivation potential of the bait LEU2 Since LEU is the reporter used in the initial screen it is important not to have a high background of colonies arising due to activation of the LEU2 gene by the bait alone Also for some baits the LEU2 reporter in EGY48 is more sensitive than the lacZ reporter on pSH18 34 Therefore the ability of the bait to autoactivate the LEU2 reporter should be tested before performing a large screen a Using a sterile wooden applicator stick transfer a colony of EGY48 containing both the
12. elective medium 1 7 g yeast nitrogen base w o amino acids 5 g ammonium sulfate 0 6 g his ura trp leu dropout mix 20 g glucose or 20 g galactose 10 g raffinose for gal raff media 20 g agar if for plates Add 1 liter of distilled water and autoclave for 20 minutes For plates cool to 50 C before pouring c other YNB selective media Add the following amounts of reagents to the YNB ura his leu trp medium described above before autoclaving to make the appropriate medium Filter sterilize and store at 4 C microwave briefly if a precipitate forms trp 10 ml of 4 mg ml stock per liter of medium 0 04 mg ml final conc ura 5 ml of 4 mg ml stock per liter of medium 0 02 mg ml final conc leu 15 ml of 4 mg ml stock per liter of medium 0 06 mg ml final conc his 5 ml of 4 mg ml stock per liter of medium 0 02 mg ml final conc For example to make medium lacking only leucine add 5 ml of 4 mg ml uracil 10 ml of 4 mg ml tryptophan and 5 ml of 4 mg ml histidine to I liter of YNB ura his leu trp medium d Yeast selective X gal media 2 Bacterial amino acid solution as per c above 0 6 g his ura trp leu dropout mix 6 7 g yeast nitrogen base without amino acids 20 g glucose or 20 g galactose 10 g raffinose for gal media 20 g agar 900 ml distilled water Autoclave and cool to 65 C In a separate bottle autoclave 7 g of sodium phosphate dibasic and 3 g of sodium phosphate monobasic
13. ests Therefore although we present you with the protocol for doing a large scale screen you may first want to do a medium scale screen maybe one fifth the size of a large scale screen before performing a large scale screen D Testing Bait s ability to enter the nucleus and bind LexA operators The plasmid pJK101 contains a lacZ reporter gene whose expression is driven by the yeast GAL1 promoter However two LexA operators have been placed between the GAL1 promoter and the lacZ gene LexA fusion proteins will bind to these operators and decrease the level of GAL 1 driven lacZ expression Repression Assay a Do the following transformations into EGY48 see protocol under section VI B 1 pEG202 Bait pJK101 test 2 pRFHM1 pJK101 repression 3 pJK101 alone no repression Note Plate transformations 1 and 2 above onto YNB glu his ura and transformation 3 onto YNB glu ura plates 17 b Streak 4 colonies from each of plates 1 and 2 above onto YNB gal his ura X gal plates and 4 colonies from plate 3 onto YNB gal ura X gal plates Afetr 12 24 hours at 30 C it should be evident whether or not the bait fusion can bind to the LexA operators If some level of repression is observed then you can proceed with the screen If no repression is seen then you should test whether the bait is even being made you can do a Western blot if antibodies against your bait exist If you see evidence that the protein is bei
14. il colonies appear 11 Calculate the number of transformants obtained by counting the number of colonies on the 100 mm plate 100 colonies on the plate corresponds to an efficiency of 10 ug so 30 ug x 10 ug 3 x 10 total transformants Similarly 50 colonies on the 100 mm plate corresponds to 5 x 10 ug or 1 5 x 10 total transformants A saturating screen of a mammalian library requires at least 2 x 10 transformants 12 Harvest the transformants as follows a Soak a microscope slide in ethanol then air dry b Pipet 10 ml of sterile distilled water onto each 24 cm x 24 cm plate scrape off the colonies with the long edge of the microscope slide taking care to use good sterile technique and pipet the slurry into a sterile disposable centrifuge tube c Centrifuge 5 minutes at 1500 x g 2 5K rpm in an HL 4 rotor at room temperature Pour off the supernatant resuspend the pellet in 19 a total of about 75 ml of sterile distilled water spin again as above and pour off the supernatant Resuspend the pellet in an equal volume of sterile distilled water Estimate the total volume add half a volume of sterile 50 glycerol mix and freeze 1 ml aliquots at 70 C Frozen stocks will remain good for at least 1 year when stored at 70 C F Screening for potential positive transformants 1 Titer the number of viable cells a Thaw an aliquot of the frozen yeast transformants and dilute 1 10 with YNB gal his ura trp medium
15. l enter the yeast nucleus and bind LexA operators II List of components additional materials required A LIST OF COMPONENTS NOTE You will not need all of the components listed below Read the manual carefully to determine which components will best suit your needs Yeast strains EGY48 EGY 194 EGY 188 EGY40 MAT 0 trp his3 ura3 leu2 6 LexAop LEU2 high sensitivity MAT 0 trp his3 ura3 leu2 4 LexAop LEU2 medium sensitivity MAT trp his3 ura3 leu2 2 LexAop LEU2 low sensitivity MAT 0 trp his3 ura3 leu2 0 LexAop LEU2 negative control RFY206 MATa trp1A hisG his3A200 ura3 52 lys2A201 leu2 3 mating strain Streak on YPD plates and grow at 30 C for 2 3 days start cultures from single colonies Reporter gene lacZ plasmids 2 each pSH18 34 URA3 2 um Ap 8 ops lacZ high sensitivity pJK103 URA3 2 um Ap 2 ops lacZ medium sensitivity pRB1840 URA3 2 um Ap 1 op lacZ low sensitivity pJK101 URA3 2 um Ap GAL1 2 ops lacZ used in repression assay Bait plasmids pEG202 HIS3 2 um Ap constitutive ADH promoter expresses LexA and is followed by a polylinker for making the bait fusion protein 10 ug pEG202 NLS HIS3 2 um Ap similar to pEG202 but with SV40 nuclear localization sequence between LexA and polylinker 2 ug pNLexA HIS3 2 um Ap similar to pEG202 except that the LexA sequence is 3 rather than 5 of the polylinker 2 ug Target plasmid pJG4 5 TRP1 2 um
16. l of solution v 1 ml of solution vi and 0 5 ml of solution vii Mix well and pour plates immediately 11 50 ml of 1 M tris pH 7 5 0 1 M 10 ml of 0 5 M EDTA 0 01 M Add 440 ml distilled water and autoclave for 20 minutes 10 X LiOAc 51 g of lithium acetate 1 M Add 500 ml of distilled water mix and autoclave for 20 minutes 50 PEG 3350 250 g polyethylene glycol 3350 Add 500 ml of distilled water mix until dissolved and autoclave for 20 minutes 1 X TE LiOAc Right before use mix 1 part 10 X TE 1 part 10 X LiOAc and 8 parts sterile distilled water 1 X TE LiOAc PEG Right before use mix part 10 X TE 1 part 10 X LiOAc and 8 parts 50 PEG 3350 Z buffer 16 1 g of sodium phosphate dibasic 60 mM 5 5 g of sodium phosphate monobasic 40 mM 0 75 g of potassium chloride 10 mM 0 246 g of magnesium sulfate I mM 2 7 ml of B mercaptoethanol 50 mM Dissolve in 1 liter of distilled water DO NOT AUTOCLAVE 7 100 mg ml X gal Dissolve 100 mg of X gal in I ml of N N dimethyl formamide and store at 20 C 12 8 Plasmid rescue solution 85 ml of distilled water 2 ml of triton X 100 2 10 ml of 10 SDS 1 2 ml of 5 M sodium chloride 0 1 M 1 ml of 1 M tris pH 8 0 0 01 M 0 2 ml of 0 5 M EDTA 0 001 M Mix and store at room temperature 9 3M sodium acetate 40 8 g of sodium acetate trihydrate Dissolve in 100 ml of distilled water and autoclave for 20 minutes
17. mid re streak or replica the colonies from the LBA plate to a minimal trp plate Grow at 37 C overnight j Do minipreps on at least 2 colonies from each plate since more than one target plasmid can get into a particular yeast cell Digest with EcoRI Xhol to release the insert and run on a gel to check the size I Mating tests of potential positives To test whether the potential positives are specific for the particular bait used you should test them against other baits with which they should not interact This is most easily done by transforming your recovered library plasmid back into the yeast strain you used in the screening EG Y48 for example transforming pSH18 34 a test bait plasmid into RFY206 and mating the two strains Example Using the small scale transformation protocol transform EG Y48 with your isolated potential positive target plasmid Similarly transform RFY206 with pLexA Max pSH18 34 Mate some transformants from each plate by streaking the two in a pattern on a YPD plate 25 EGY48 FY206 Incubate the plate at 30 C overnight and replica to a YNB gal his ura trp plate the next day Grow at 30 C for 1 2 days The only cells that should grow are the ones at the intersection of the two streaks Perform lacZ filter assays to test for lacZ expression pLexA Max pBait and pRHFM1 are all bait plasmids that should not interact with your target You should also test your target against the
18. ng made in yeast then try making your bait construct in pEG202 NLS formerly called pJK202 see Appendix pEG202 NLS contains a nuclear localization sequence just upstream of the pEG202 polylinker E Large scale library screen protocol 1 Grow a 20 ml overnight culture at 30 C of EGY48 or EGY 194 or EGY 188 containing the lacZ reporter plasmid pSH18 34 for example and your bait plasmid Grow in YNB glu his ura medium 2 The next morning dilute 100 ul of culture into 0 9 ml of water mix well and immediately measure the OD of the dilution Multiply by 10 to get the OD of the undiluted culture Inoculate 300 ml of YNB glu his ura in a sterile 2 L flask with enough of the overnight culture to give an OD 0 1 For example if the dilution of the overnight culture had an OD 0 5 then the 20 ml undiluted overnight culture would have an ODs 0 5 x 10 5 Since you want the 300 ml culture to start at OD 0 1 the amount of undiluted culture needed would be 0 1 5 x 300 ml 6 ml Therefore you would add 6 ml of undiluted overnight culture to 300 ml of YNB glu his ura and grow at 30 C with vigorous shaking Note Without agitation yeast cells will settle to the bottom of a flask over time so you should always swirl the flask before removing any culture to ensure the culture is of uniform density 3 Shake the culture vigorously at 30 C until the OD 0 5 0 7 This should take 4 5 hours 4 Harvest the cells by spin
19. ning at 1500 x g 3K rpm in a GSA rotor for 5 minutes room temperature Pour off the supernatant 5 Resuspend in 30 ml of sterile distilled water transfer to a 50 ml sterile conical tube and spin at 1500 x g 2 5K rpm in an HL 4 rotor for 5 minutes room temperature Pour off the supernatant 18 6 Resuspend the cell pellet in 1 5 ml of 1 x TE LiOAc Aliqout 50 ul portions into 30 sterile 1 5 ml eppendorf tubes Note A better transformation efficiency is obtained when doing several small transformations rather than one large transformation 7 Add 50 ug 10 ul of carrier DNA and 1 ug of pJG4 5 based plasmid library DNA to each eppendorf tube Do not use more than I ug of library DNA per tube since multiple plasmids can enter the same yeast cell and give confusing results in later analyses 8 Add 300 ul of I x TE LiOAc PEG to each tube and mix by inversion Incubate without agitation at 30 C for 30 minutes 9 Add 40 ul of DMSO to each tube mix by inversion and heat shock by incubating at 42 45 C for 10 minutes 10 Add 0 6 ml of sterile distilled water to each tube and mix by inversion Dilute 10 ul of one tube into 990 ul of sterile distilled water vortex and plate 100 ul of the dilution onto a 100 mm YNB glu his ura trp plate Plate all of each tube onto 30 separate 24 cm x 24 cm YNB glu his ura trp plates or 300 ul onto each of 100 150mm YNB glu his trp ura plates Incubate all plates at 30 C for 2 3 days unt
20. o another master plate for colonies which arise three days after plating and another master plate for colonies which arise four days after plating Colonies from the first two master plates should definitely be characterized further Colonies that do not appear until about a week after plating are likely to be artifactual and should not be characterized further unless they are the only colonies obtained 3 Test galactose growth dependence and lacZ expression of potential positive transformants a Since the expression of the target protein is dependent on galactose any colonies which can activate LEU2 and grow on leu medium in the presence of glucose are false positives and should not be further characterized Colonies that can grow on leu medium containing galactose but that cannot grow on leu medium containing glucose are potentially true positives and should be tested for lacZ expression Re streak colonies from the master plates to the following types of plates YNB glu his ura trp leu YNB gal his ura trp leu YNB glu his ura trp X gal YNB gal his ura trp X gal b Incubate at 30 C for 1 2 days until growth occurs Potential positive transformants will grow on the leu gal plate but not the leu glu plate and will turn blue on the X gal gal plate but not on the X gal glu plate c If the number of potential positives is small lt 50 then all should be recovered and further characterized If gt 50 potential posi
21. onal activation can be detected If binding sites for Gal4p are placed upstream of a reporter gene such as lacZ transcriptional activation can be monitored easily The DupLEX A system utilizes the same basic idea except that the DNA binding protein is the E coli LexA protein while the activation protein is the acid blob domain B42 Neither LexA protein bound upstream of a reporter gene nor B42 alone can activate transcription of the reporter but if brought together via fusions with two interacting proteins reporter gene expression can be detected Advantages of the DupLEX A system over other yeast two hybrid systems include reduction in the number of false positives obtained since prokaryotic LexA and B42 rather than eukaryotic Gal4p proteins are used ability to screen potentially toxic target proteins since their expression is galactose inducible ability to demonstrate a potential positive s interaction with bait is dependent upon expression of the potential positive ease of doing a coimmunoprecipitation assay of bait and potential positive since antibodies to HA tag fused downstream of B42 are available if antibody to the bait protein is also available reporters with varying sensitivities are available so that baits which activate transcription on their own can potentially still be assayed simply by using a less sensitive reporter ease of determining whether or not a particular bait protein wil
22. sing standard recombinant DNA techniques subclone your bait protein gene in the correct orientation into the polylinker of pEG202 see Appendix Design the bait protein gene subcloning such that it fuses in frame with LexA We strongly recommend verifying the sequence of the LexA bait junction to make sure that a LexA cDNA fusion protein should be made NOTE We highly recommend testing your bait fusion protein in the assays below before performing a full scale library screen B Testing the autoactivation potential of the bait lacZ Some bait proteins can activate reporter genes on their own making a two hybrid system library screen a waste of time effort and resources However the DupLEX A system offers some alternatives if this occurs To test for autoactivation by your bait fusion transform yeast strain EGY48 with the following combinations of vectors 1 pEG202 Bait pSH18 34 test 2 pSH17 4 pSH18 34 strong activation 3 pRFHM1 pSH18 34 no activation 14 Small scale Yeast Transformation Protocol a Grow a5 ml culture of EG Y48 in YPD at 30 C with shaking overnight Inoculate by picking a colony off of a streaked plate of EGY48 b Measure the OD of a 1 10 dilution of the overnight culture Calculate the OD of the 5 ml culture and use that to inoculate a 60 ml YPD culture to an OD 0 1 Grow at 30 C with shaking c When the OD600 0 5 0 7 approximately 4 6 hours after inoculation pelle
23. smid DNA Also the trp E coli strain used KC8 is not very amenable to transformation Therefore electroporation is recommended to recover the library plasmid by transforming into KC8 cells Making electrocompetent KC8 cells a Streak KC8 onto a LBK plate and grow at 37 C overnight b Pick a single colony and inoculate into 5 ml of LBK medium Grow at 37 C overnight with shaking c Use all 5 ml to inoculate a 500 ml culture in LBK medium Grow with shaking at 37 C until the OD 0 5 d Chill the cells on ice for 30 minutes Spin at 5 K rpm for 10 minutes at 4 C 22 e Pour off the supernatant and resuspend the pellet in 300 ml of ice cold 10 glycerol Spin again as in d above pour off the supernatant and resuspend the pellet in 150 ml of ice cold 10 glycerol f Spin again as in d above pour off the supernatant resuspend the pellet in 10 ml of ice cold 10 glycerol spin again as in d above carefully pipet off and discard the supernatant and resuspend the pellet in 2 ml of ice cold 10 glycerol g Aliquot into 75 ul portions and store at 70 C h Electroporate 1 ul of the recovered DNA from step VI G 4 above into 75 ul of competent KC8 cells using the specifications for your particular electroporator Plate onto LBA plates and grow at 37 C overnight Colonies arising at this stage contain either the bait target or lacZ reporter plasmid i To select only those colonies which contain the target plas
24. t the cells by spinning the culture at 1500 x g for 5 minutes Resuspend in 20 mls of sterile distilled water spin again and resuspend the pellet in 0 3 ml of 1 x TE LiOAc Put 100 ul into each of three sterile 1 5 ml eppendorf tubes d Add 100 ng of each plasmid DNA and 50 ug of carrier DNA to each tube and mix e Add 0 3 ml of 1 x TE LiOAc PEG mix by inversion and put the tubes at 30 C with or without shaking for 30 minutes f Add 70 ul of DEMSO dimethyl sulfoxide to each mix by inversion and put at 42 45 C without shaking for 15 minutes g Spin at 1OK rpm in a microcentrifuge for 10 seconds pour off the supernatant and resuspend each pellet in 0 5 ml of sterile distilled water h Spread 50 100 ul of each onto separate YNB glu his ura plates Incubate at 30 C for 2 3 days Streak 4 colonies from each plate onto another YNB glu his ura plate Inoculate at 30 C 1 2 days Perform a lacZ filter assay or replica to YNB gal his ura X gal plates and grow at 30 C overnight 15 Filter Assay Cut a piece of Whatman 3M paper such that it just fits into a 100 mm petri dish Put it in an empty dish and add 2 ml of 1 mg ml X gal add 20 ul of X gal in N N dimethyl formamide to 2 ml of Z buffer maikng sure the filter is completely wet Place a similarly cut nitrocellulose filter on the surface of the plate containing the re streaked yeast then pull off and freeze at 70 C for 5 minutes Take out thaw and r
25. tives are obtained then you should chracterize the first 50 that arise and freeze the rest at 70 C in 20 glycerol G Recovering potential positive plasmids from yeast 21 1 To isolate DNA from the potential positives grow each one in 2 ml of YNB glu trp medium or any other trp medium you have available at 30 C overnight 2 Spin down 1 5 ml of each in eppendorf tubes for 10 seconds at maximum speed Pour off the supernatant vortex to resuspend the pellet in the residual liquid and add 200 ul of plasmid rescue solution 3 Add 100 ul of phenol tris sat pH 8 0 and 100 ul of 24 1 chloroform isoamyl alcohol Add about 0 3 g of acid washed glass beads and vortex vigorously for 2 minutes 4 Spin at 14 K rpm in a microcentrifuge for 5 minutes at room temperature Carefully remove 200 ul from the top aqueous layer and transfer it to a clean eppendorf tube Add 20 ul of 3M NaOAc vortex and add 440 ul of 95 ethanol Vortex spin 20 minutes at 14 K rpm in a microcentrifuge pipet off and discard the supernatant wash with 70 ethanol carefully pipet off and discard the supernatant vacuum dry the pellet and resuspend the pellet in 5 ul of sterile distilled water Use 1 ul to transform E coli KC8 cells to TRP see below H Obtaining potential positve library plasmids in bacteria 1 The pellet recovered from the yeast cells in the above procedure consists mostly of yeast RNA and genomic DNA Very little of it is library pla
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