User`s manual - Boca Scientific Laboratory Products
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1. Add 340 pl of 1x TE LiOAc PEG mix by inversion and put the tubes at 30 C with or without shaking for 30 minutes i Incubate at 42 C without shaking for 15 minutes i Add 600 yl sterile distilled water spin at 1500 g in a microcentrifuge for 5 minutes pour off the supernatant and resuspend each pellet in 100 pl of sterile distilled water k Dilute 10 pl of each transformation suspension in 990 pl sterile distilled water and spread 100 pl of this dilution onto separate appropriate selective DOBA plates e g HIS or URA Incubate at 30 C for 2 3 days Calculate the number of transformants obtained by counting the number of colonies on the plate 100 colonies on the plate corresponds to an efficiency of 2 x 104 pg plasmid DNA when transforming with e g 250 pg of bait and 250 pg of prey plasmid Streak 4 colonies from each plate onto another DOBA glu HIS URA plate Incubate at 30 C 2 3 days Perform a GFP assay with a UV lamp in a darkroom 01 2001 gt 25 MoBi Tec Appendix Il Large Scale Yeast Transformation Procedure a Grow a 30 ml culture of yeast in YPD medium at 30 C with shaking for 24 hours Inoculate by picking a colony from a streaked ITH5 plate b Measure of a 1 10 dilution of the overnight culture Calculate the OD of the 30 ml culture and use that to inoculate 2 x 500 ml culture to an OD 0 1 Grow at 30 C with vigorous shaking Once the2. Alternatively you can co transform yeast strain ITH5 with the bait vector carrying your target gene and the empty prey vector Clones growing on medium containing CuSO and or fluorescing green under UV light are not suitable for a two hybrid screen Clones which do not grow on CuSO containing medium and which do not fluoresce green can be further tested for interaction of a bait and a prey protein in a two hybrid screen For this purpose the bait vector and the prey vector e g a cDNA library are transformed into yeast If the expressed bait and prey proteins are interacting the DNA binding and transactivation domains of ACE 1 are tethered to one another and transactivate the reporter genes CUP and GFP both integrated in the yeast chromosome 01 2001 46 MoBi Tec 2 Schematic Overview of the Grow n Glow System 1 TEST FOR AUTOACTIVATION BY BAIT Yeast strain with chromosomal CUP1 and GFP reporter genes transformed with bait vector ACE1BD bait no autoactivation no transcription l GFP or CUP1 reporter gene suited for two hybrid screen autoactivation ey transcription green fluorescence growth on selective copper containing medium not suited 2 1 GFP or CUP1 reporter gene for two hybrid screen 2 TEST FOR INTERACTION OF BAIT amp PREY Yeast strain bait prey with chromosomal vector vector CUP1 and GFP reporter genes transformed with ACE1BD bait
3. for some baits the reporter in ITH5 may be more sensitive than the GFPuv reporter Therefore the ability of the bait to autoactivate the CUP1 reporter should be tested before performing a large screen To test for autoactivation by your bait fusion protein transform yeast strain ITH5 with the bait vector containing your bait gene in the correct reading frame 7 2 1 Yeast Transformation Protocol We recommend the Grow n Glow High Efficiency Yeast Transformation Kit order 2200 1 However for the above described testing of autoactivation other transformation procedures can be used as well see Appendix 1 a Use the protocol supplied with the Grow n Glow High Efficiency Yeast Transformation Kit using 200 ng of plasmid DNA for transformation or the procedure described in Appendix up to step b Spread 50 100 pl of each transformation onto separate DOBA glu HIS plates c Incubate at 30 C for 2 3 days Streak 4 colonies from each plate onto another DOBA glu HIS plate e Incubate at 30 C 1 2 days f Perform a CUP1 autoactivation assay as follows see 7 2 2 7 2 2 Testing Procedure for CUP1 Autoactivation a Transfer a colony of ITH5 containing the bait plasmid into 0 5 ml of sterile distilled water 01 2001 gt 17 MoBi Tec b Vortex c Dilute 100 pl into 1 ml of sterile distilled water d Vortex this is Dilution 1 e Do three more serial 1 10 dilutions Dilutions 2 4 suc
4. transcription green fluorescence growth on selective copper containing medium CUP1 regulat CUP1 promoter GFP or CUP1 reporter gene Legend see page 6 ACE1AD prey e g a cDNA library no interaction of bait and prey ACE1BD and ACE1AD are separated interaction of bait and prey ACE1BD and ACE1AD are teth ered to one another and transactivate the reporter gene 01 2001 gt MoBi Tec 3 Advantages of the Grow n Glow ACE1 System interaction is detected directly by visualisation under UV light allows to select for different protein interaction strengths reat reduces amount of time amp effort needed to screen a cDNA ibrary allows immediate inspection of yeast colonies for gene activation non invasive chemical free and cost free assay e stable GFP expression GFP may be used as sole reporter gene no requirement for external substrates higher threshold for detection of protein protein interactions fewer false positives by stringent selection for copper resistance growth selection can be modulated by different CuSO concentrations suited for the development of high throughput screening assays 4 Kit Components Complete Kit The Grow n Glow ACE Two Hybrid System is offered as a Complete Kit for scientists starting to establish the two hybrid technology Grow n Glow Two Hybrid System Complete Kit Vectors Primers 137 5 yg 5 BAlTprimer 5
5. OD 0 5 0 7 approximately 4 6 hours after inoculation pellet the cells by spinning the culture at 1500 x g for 5 minutes Resuspend in 2 x 50 ml of sterile distilled water transfer to two 50 ml sterile conical tubes spin again and resuspend the two combined pellets in 20 ml of 1 x TE LiOAc d Spin again at 1500 g for 5 minutes and resuspend the pellet in 5 ml of 1 x TE LiOAc Boil the carrier DNA 3 times for 5 minutes and quickly chill on ice This is essential for obtaining a maximum efficiency of transformation f Add 500 ng of each plasmid DNA to 25 yg of denatured carrier DNA 10 pl and mix For large scale transformation of yeast cells containing already bait plasmid DNA pTY137 insert with library plasmid DNA use 1 pg of pTM114 based plasmid library DNA and 50 yg of carrier DNA Do not use more than 1 pg of library DNA per tube since multiple plasmids can enter the same yeast cell and give confusing results in later analyses g Add 50 pl resuspended cells from step d to the DNA 10 pl from step f h Add 340 pl of 1x TE LiOAc PEG mix by inversion and put the tubes at 30 C with gentle or no agitation for 30 minutes i Incubate at 42 C without shaking for 15 minutes i Add 600 pl water to each tube spin at 1500 g in a microcentrifuge for 5 minutes pour off the supernatant and resuspend each pellet in 100 pl of sterile distilled water k Dilute 10 pl of each transformation suspension in 990 pl st
6. Tec 24 14 15 16 Crameri A E A Whitehorn E Tate and W P C Stemmer 1996 Improved green fluorescent protein by molecular evolution using DNA shuffling Nature Biotechnol 14 315 319 Chalfie M Y Tu G Euskirchen W W Ward and D C Prasher 1994 Green fluorescent protein as a marker for gene expression Science 263 802 805 Chiocchetti A E Tolosano E Hirsch L Silengo and F Altruda 1997 Green fluorescent protein as a reporter of gene expression in transgenic mice Biochim Biophys Acta 1352 193 202 Yeh E K Gustafson and G L Boulianne 1995 Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila Proc Natl Acad Sci USA 92 7036 7040 Meng Tang A Ong J Farrell and S Lin 1997 Promoter analysis living zebrafish embryos identifies a cis acting motif required for neuronal expression of GATA 2 Proc Natl Acad Sci USA 94 6267 6272 Cormack R S Hahlbrock and I E Somssich 1998 Isolation of putative plant transcriptional coactivators using a modified two hybrid system incorporating a GFP reporter gene Plant J 685 692 Niedenthal K L Riles M Johnston and J H Hegemann Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast Yeast 12 773 786 Sambrook J E F Fritsch and T Maniatis 1989 Molecular Cloning A Laboratory Manual Cold Spring H
7. Tec DOB HIS glucose Pour the entire content of a DOB glucose bag order 4025 1 and 0 385 g CSM HIS order 4510 3 into a 0 5 flask add 500 ml H O Autoclave Cool to at least 37 C Ready for use DOB URA HIS glucose Pour the entire content of a DOB glucose bag MoBiTec order 4025 1 and 0 375 g CSM URA HIS MoBiTec order 4520 3 into a 0 5 flask add 500 ml H O Autoclave Cool to at least 37 C Ready for use DOBA HIS glucose Pour 21 85 g DOBA glu MoBiTec order 4026 1 and 0 385 g CSM HIS MoBiTec order 4510 3 into a 0 5 flask add 500 ml H O Autoclave Cool to 55 C Pour into plates Ready for use DOBA URA glucose Pour 21 85 g DOBA glu MoBiTec order 4026 1 and 0 385 g CSM URA order 4511 2 into a 0 5 flask add 500 ml H O Autoclave Cool to 55 C Pour into plates Ready for use DOBA URA HIS glucose Pour 21 85 g DOBA glu MoBiTec order 4026 1 and 0 375 g CSM URA HIS MoBiTec order 4520 3 into a 0 5 flask add 500 ml H O Autoclave Cool to 55 C Pour into plates Ready for use 5 1 2 Grow n Glow Bacterial Growth Media a LB Medium Pour the entire content of an LB medium bag MoBiTec order 3002 1 into 0 5 flask add 500 ml H O Autoclave Cool to at least 37 C Ready for use b LB Agar Medium Pour the entire content of an LB agar medium bag order 3002 2 into a 0 5 flask add 500 ml
8. for 15 minutes It is very importantto keep the temperature at 0 C unless otherwise stated d Pellet cells by centrifugation Spin 10 minutes at 6 000 rpm 3 000 at 4 C e Discard the supernatant f Resuspend in 80 pl ice cold RF I buffer see below g Place on ice for 20 minutes h Spin 6 000 rpm for 10 minutes at 4 C i Discard the supernatant Resuspend the pellet in 20 ml ice cold RF Il buffer see below i Aliquote the cells in 200 pl aliquots Eppendorf tubes on ice Quick freeze immediately in liquid nitrogen and place at 80 C until use Materials SOB 2 w v Bacto Tryptone 0 5 w v yeast extract 10 mM NaCl 2 5 mM KCI 10 mM MgCl 10 mM MgSO 01 2001 gt 29 MoBi Tec 30 RF I buffer RF Il buffer 100 mM RbCl rubidiumchloride 30 mM KAc pH 7 5 10 mM CaCl 15 w v glycerol 87 Adjust to pH 5 8 with 0 2 M acetic acid Add 50 mM MnCl 10 mM MOPS 10 mM RbCl 75 mM CaCl 15 w v glycerol 87 Adjust to pH 6 8 with 1 M NaOH Transformation Procedure a Thaw the frozen competent cells at 4 C and store on ice b Add plasmid DNA c Incubate on ice for 30 60 minutes d Incubate in a water bath preheated to 42 C for exactly 2 minutes f Add 1 ml LB medium g Incubate at 37 C for one hour h Plate the transformation solution onto selective media LB medium supple mented with 100 pg ml ampicillin i Incubate overnight or 8 16 hou
9. genetic and molecular biological methods due to its ability to be transformed by foreign DNA and its highly efficient system of homologous recombination Note that binding of the ACETBD to DNA is dependent on the presence of copper ions Thus for selection of clones expressing interacting proteins media has to be supplemented with CuSO 2 10 pM 7 Grow n Glow Two Hybrid System Protocol 7 1 Constructing the Hybrid Gene ACE1BD Bait Using standard recombinant DNA techniques subclone your bait protein gene in the correct orientation into the polylinker of pTY137 see chapter 4 2 1 Design the subcloning of the bait gene such that it fuses in frame with ACE1 BD 01 2001 t MoBi Tec We strongly recommend verifying the sequence of the ACE1BD bait junction with the sequencing primer 5 BAlTprimer provided in the Complete Kit to make sure that a ACE1BD bait fusion protein is going to be expressed correctly Note We highly recommend testing your bait fusion protein in the assays below before performing a full scale screen of e g a cDNA library constructed in the prey vector 7 2 Autoactivation of A two hybrid system library screen does not have to be performed if the boit protein activates the reporter genes on its own autoactivation Since CUP1 is the reporter used in the initial screen it is important not to have a high background of colonies arising due to autoactivation of the gene Also
10. into the yeast strain simultaneously or sequentially We recommend the following strategy a First introduce the bait plasmid and plate onto selective DOBA glu HIS plates b One yeast colony from the DOBA glu HIS plates is re streaked onto a DOBA HIS plate One colony is then selected and propagated for transformation with the prey library or one prey plasmid with your insert of interest c Dilute 100 pl transformation suspension see Appendix II in 900 pl sterile distilled water and spread the diluted suspension onto 10 DOBA glu HIS URA plates 150 mm supplemented with 30 pM CuSO Spread serial dilutions of the transformation suspension onto a DOBA glu HIS URA plate without CuSO to determine the transformation efficiencies see Appendix III k l Note The transformation of the yeast strain with pTY 137 is performed as small scale transformation and the libra ry transformation as a large scale transforma tion 7 5 Green Fluorescent Protein GFP Assay Protocol The copper containing DOBA glu HIS URA plates grown with the putative positive colonies are exposed to standard UV light in a darkroom The colonies showing green fluorescence are putative most probably positive These colonies are isolated by streaking them onto new DOBA glu HIS URA plates supplemented with CuSO master plate and by incubating them for 24 72 hours at 30 C until colonies appear which are then checked again for GFPuv expr
11. n Glow High Efficiency Yeast Transformation Kit MoBiTec order 2200 1 or alternatively Grow n Glow Fast and Easy Yeast Transformation Kit MoBiTec order 2100 1 5 Grow n Glow Yeast Plasmid Isolation Kit MoBiTec order 2069 1 5 1 Recipes for Media For optimal results we highly recommend to use the Grow n Glow Yeast and Bacterial Growth Media offered by MoBiTec see chapter 5 1 1 and 5 1 2 which are optimized for two hybrid systems Rich medium YPD and the standard Wickerham yeast nitrogen base with carbon source optimized for S cerevisiae can be added to water and autoclaved without the need to make concentrated solutions of vitamins trace elements salts or carbon sources The powder dropout base formulation which is called DOB Drop Out Base medium or DOBA Drop Out Base with Agar is a complete standard Wickerham yeast nitrogen base with a carbon source A complete supplemented synthetic defined medium is easily made by mixing two powders DOB or DOBA and CSM Complete Supplement Mixture The formulation of CSM is a dropout supple ment for virtually all strains of S cerevisiae containing different combinations of amino acids adenine and uracil Cells grow vigorously in DOB supplemented with CSM These media are very easy to handle and are delivered as powder Some of our media are available in small bags with the appropriate amount of powder for 0 5 litre medium 10 bags per package are sufficient for 10 x 0 5 litre m
12. tar get protein Fusion protein ex pression is controlled by the con stitutive Acel promoter Acel p For selection in yeast the vector contains the HIS3 histidine marker and the ars cen origin of replication For propagation in E coli an ampicillin resis tance Amp is present CYC1t terminator sequences of the cy tochrome c1 oxidase gene pTY137 polylinker Xbal Spel BamH Sma 1 EcoR EcoRV Cla 1 Sal Hind 1 Pst AGG TCT AGA ACT AGT GGA TCC CCC GGG CTG CAG GAA TTC GAT ATC AAG CTT ATC GAT ACC GTC GAC Xbal Spel BamH Smal EcoR EcoRV Cla I Sal Polylinker of the vector indicating the open reading frame The shown restriction endonucleases only cutting the polylinker single cutters 4 2 2 Prey Plasmid pTM114 The prey vector 1 14 is used to express cDNAs or other coding sequences inserted into the mul tiple cloning site as translational fusions to a cassette consisting of the SV40 nuclear localization se quence NLS and the ACE acti vation domain Fusion protein ex pression is controlled by the strong constitutive glyceraldehyde 3 phosphate dehydrogenase GAPDH promoter For selection in yeast the vector contains the URA3 selectable marker and the 2 ym origin of replication for propagation in E coli an ampicillin resistance Sall Nco Sma EcoR NLS 2um ori pTM114 7518 bp ACE1AD Ball Spel Sac Il Pvu
13. 00 pmole pIMI 14 5 5 PREYprimer 500 pmole pTY143 5 yg 3 PREYprimer 500 pmole Ug 01 2001 t MoBi Tec Host Strain Yeast strain ITH5 1 ml Available on request not included in the kit pre transformed strains ITH11 ITH5 transformed with pTY137 and 114 ITH16 ITH5 transformed with pTY143 and pTM11 4 ITH17 ITH5 transformed with pTY137 and pTM125 ITH12 ITH5 transformed with pTY143 and pTM125 ITH15 ITH5 transformed with pTY139 Before use redissolve plasmid DNA in TE buffer pH 8 Order information see chapter 10 NOTE You may not require all ofthe components listed below Read the manua carefully to determine which components will best suit your needs 4 1 Yeast Strains The provided yeast strain has the following genotype ITH5 MATa ura3 52 his3 171 trp1 289 acel TRP1 leu2 3 112 LEU2 pTY99 The strain contains a CUP1 gene cassette multiple copies of the CUP1 gene as well as the GFP reporter gene under control of the Cup promoter stably integrated on its chromosome The CUP1 gene cassette allows growth selection via activation by ACEIBD ACE1AD as well as a test autoactivation of a reporter gene by the bait protein See chapter 6 for growth and maintenance of yeast 01 2001 gt 9 MoBi Tec 4 2 DNA Vectors 4 2 1 Bait Plasmid pTY137 The bait vector pTY137 is used to generate fusions of the binding domain with a bait
14. 6 7 Grow n Glow Two Hybrid System Protocol 16 7 1 Constructing the Hybrid Gene ACETBD Bait 16 7 2 Autoactivation of CUP T 17 7 2 1 Yeast Transformation Protocol sss 17 7 2 2 Testing Procedure for CUP1 Autoactivation 17 7 3 Autoactivation of GPP 18 7 4 Large Scale Library Screen 19 7 5 Green Fluorescent Protein GFP Assay Protocol 20 7 6 Further Characterization of Putative Positives 21 7 6 1 Plasmid Isolation from Yeast 21 7 6 2 Transformation of E coli with Plasmids Isolated from Yeast 21 7 6 3 Prey Plasmid Identification by PCR 22 7 7 Other Verification Methods of Protein Protein Interaction 23 8 literat re bv cie uno dco qaia A EE 29 O P ep tentia Ead 25 Appendix I Small Scale Yeast Transformation Procedure 25 Appendix II Large Scale Yeast Transformation Procedure 26 Appendix Ill Isolation of Plasmids from Yeast 28 Appendix IV Transformation of competent E coli Cells 29 10 Order Information Shipping and Storage 31 01 2001 46 2 MoBi Tec The Grow n Glow ACE1
15. 7 and test for a part that does not autoactivate Once you are convinced that your bait fusion can bind to CUP1 regulatory sequences within the promoter without autoactivating either of the two reporter genes then you are ready to perform a large scale library screen 7 4 Large Scale Library Screen Protocol This step is very critical Use standard recombinant DNA techniques to construct your cDNA library in the prey vector Clone the cDNA fragments in the correct orientation into the polylinker of pTM114 see chapter 4 2 3 Design the subcloning of the cDNA fragments such that they fuse in frame with ACE1AD to make sure that ACE1AD prey fusion proteins are going to be expressed correctly The number of recombinants transformed with the prey clones should be as high as possible We therefore recommend the use of the Grow n Glow High Efficiency Yeast Transformation Kit MoBiTec order 2200 1 for obtaining the best results since it contains an optimized library transformation procedure 01 2001 gt 19 MoBi Tec 20 Otherwise you can try an up scaling of the procedure as described in Appendix II with the following information To screen up to 106 independent clones you should use 100 yg library plasmid DNA for transformation of one litre yeast culture When screening a prey library to find new proteins that interact with the bait protein the bait and prey plasmids see chapter 4 2 can either be transformed
16. E1 allowing the protein to interact with DNAS The conventional reporter gene generally used with yeast two hybrid systems is p galactosidase 01 2001 46 MoBi Tec The new Grow n Glow System was developed at the Hans Knell Institute in Jena as a more versatile and more accurate version of the yeast two hybrid system since it is using the reporter gene GFP The practical advantages of this fluorescent protein over B galactosidase are substantial In a typical B Gal screen individual surviving yeast colonies are picked and then tested for galactosidase activity either by a filter assay or growth on minimal medium plates containing X Gal Both procedures are often labor intensive especially when hundreds of yeast colonies are obtained In contrast when GFP is used as the reporter the selection plates containing the yeast colonies are simply placed under a UV lamp without the lid and positives become immediately identifiable by their green fluorescence In summary the GFP gene stably integrated in the yeast chromosome is a valuable addition to the family of reporter genes for two hybrid systems and makes a secondary screening of yeast colonies faster and more cost effective than conventional LacZ assays Detection of protein protein interactions via the green fluorescent protein provides global screening of colonies without bias and can be adapted to most yeast based two hybrid strategies In addition the coppe
17. H O Autoclave Cool to 55 C Pour into plates Ready for use c LB Amp Medium ampicillin selection Cool the LB medium a above to 37 C and add 4 ml of 25 mg ml ampicillin in distilled water filter sterilized per litre of medium Mix 01 2001 gt 15 MoBi Tec d LB Amp Agar Medium ampicillin selection Cool the LB agar medium above to 55 C and add 4 ml of 25 mg ml ampicillin in distilled water filter sterilized per litre of medium Mix Pour into plates Recipes for preparing E coli growth media are described in Sambrook et al 1989 and Ausubel et al 1997 recipes for the preparation of yeast growth media in Ausubel et al 1997 and Guthrie and Fink 1991 7 6 Growth and Maintenance of Yeast The yeast strain Saccharomyces cerevisiae see chapter 4 1 in our Complete Kit are provided in YPD medium with 20 glycerol and can be maintained indefinitely at 80 C Streak the yeast strains do not thaw the strains ever on YPD plates Start cultures from single colonies and grow in YPD medium at 30 C for 2 3 days General remarks Yeast can be grown on plates or in liquid culture like E coli However antibiotics which work on E coli do not work on yeast making good sterile technique mandatory when working with yeast The optimum growth temperature for yeast is 28 32 C The growth rate is relatively rapid with a doubling time of 90 120 minutes Budding yeast is very amenable to
18. Il 1 Amp 01 2001 MoBi Tec pTM114 polylinker TGG CCA GCG GAT CCA CTA GIT CTA GAG CGG CCG CCA CCG CGG Bal BamH Spe Sac II Polylinker of the vector indicating the open reading frame The shown restriction endonucleases only cutting the polylinker single cutters 4 2 3 Control Plasmids Bait control The bait control plasmid pTY 1 43 is a derivative of pTY137 and carries the coding sequences for the N terminal dimerization part of Bcy1 gene encoding the regulatory subunit of the yeast cAMP dependent protein ki nase fused to the ACE1BD pIM143 serves as positive con trol in combination with the prey control vector pTM125 Since both control plasmids carry the coding sequences for the inter acting N terminal dimerization part of Boyl Sst Amp Acelp ACE1 BamHI CEN6 ADENA pTY143 ori CYCit Bcy1 gene EcoRI Hind Hind 11 Pstl Prey control Nco The prey control plasmid pTM125 a derivative of pTM1 14 also carries the coding sequences for the N terminal dimerization part of the Bcylgene encoding the regulatory subunit of the yeast cAMP depen dent protein kinase which is here fused to the ACE1AD Sma PTM 125 CYC1t Pvu II Pvu II BamHI N terminal part of the Bcy1 gene 01 2001 gt 11 MoBi Tec Positive control Sacl The control plasmid pTY139 can ac tivate transcription
19. Mo Bi Tec GRow N Giow THE Two Hysrip SYSTEM INFORMATION AND PROTOCOLS January 2001 Grow n GLow THe Two Hysrip SYSTEM 2001 E Mo Bi Tec GmbH lt 3 55 Lotzestrasse 22a D 37083 G ttingen F 71 49 551 707 22 0 Fax 49 551 707 22 22 E mail order mobitec de Mo Bi Tec or info mobitec de MOLECULAR BIOLOGISCHE TECHNOLOGIE INTERNET http www mobitec de MoBi Tec Content page Te MOU CHO es 4 1 1 GOCE sei cS iof ce Aoc a ud ait ded tds ede d 4 1 2 sero tetur ales 4 1 3 CORP EP EEEE 6 2 Schematic Overview of the Grow n Glow ACE1 System 7 3 Advantages of the Grow n Glow System 8 4 Components Complete 8 4 1 Yeast Strains 9 4 2 DNA Vectors 10 4 2 1 Bait Plasmids lY 10 4 2 2 Prey Plasmid DIM T IA erento te 10 4 2 3 Control Plasmids ossa rated MEE a ui UP Ui 11 4 3 Primerane oan E NOE TN 12 5 Materials Required but not Supplied 13 5 1 Recipes for Media 13 5 1 1 Grow n Glow Yeast Growth Media 14 5 1 2 Grow n Glow Bacterial Growth Media 15 6 Growth and Maintenance of 1
20. arbor Laboratories Cold Spring Harbor NY Ausubel F M R Brent R E Kingston D D Moore J G Seidmann J A Smith and K Struhl 1997 Current Protocols in Molecular Biology John Wiley and Sons Inc New York 17 Guthrie C and G R Fink 1991 Guide to yeast genetics and molecular biology Methods of Enzymology 194 01 2001 MoBi Tec 10 Appendix Appendix I Small Scale Yeast Transformation Procedure a Grow a 5 ml culture of yeast in YPD medium at 30 C with shaking for 24 hours Inoculate by picking a colony from a streaked ITH5 plate b Measure OD of a 1 10 dilution of the overnight culture Calculate the 5 ml culture and use that to inoculate a 60 ml YPD culture to an OD 0 1 Grow at 30 C with vigorous shaking Once the OD 0 5 0 7 approximately 4 6 hours after inoculation pellet the cells by spinning the culture at 1500 x g for 5 minutes Resuspend in 20 ml of sterile distilled water spin again at 1500 g and resuspend the pellet in 5 ml of 1 X TE LiOAc d Spin again at 1500 g and resuspend the pellet in 300 yl of 1x TE LiOAc Boil the carrier DNA 3 times 5 minutes and quickly chill on ice This is essential for obtaining a maximum efficiency of transformation f Add 250 ng of each plasmid DNA e g pTY 137 insertand pTM1 14 insert to 25 pg of denatured carrier DNA 10 pl and mix g Add 50 pl resuspended cells from step d to the DNA 10 pl from step f
21. bstitu tions or frame shift mutations of the gene and assaying the relative strength of the interactions using the GFP reporter assay and or growth selection on medium containing different concentrations of copper ions 8 Literature 1 Fields S and O Song 1989 A novel genetic method to detect protein protein interactions Nature 340 245 246 2 Shirley B W and 1 Hwang 1995 The interaction trap in vivo analysis of protein protein interactions Methods Cell Biol 49 401 416 3 Allen J B M W Walberg M C Edwards and S J Elledge 1 995 Finding prospective partners in the library the two hybrid system and phage display find a match Trends Biochem Sci 20 511 516 4 Cubitt A B R S R Adams E Boyd L A Gross and Y Tsien 1995 Understanding improving and using green fluorescent proteins Trends Biochem Sci 20 448 455 5 F rst P S Hu R Hackett and D Hamer 1988 Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein Cell 55 705 717 6 Mayer G H Launhardt and T Munder 1999 Application of the green fluorescent protein as a reporter for Ace based two hybrid studies BioTechniques 27 86 94 7 Munder T and P F rst 1992 The Saccharomyces cerevisiae Cdc25 gene product binds specifically to catalytically inactive Ras proteins in vivo Mol Cell Biol 12 2091 2099 01 2001 gt 23 MoBi
22. edium Just add water and autoclave ready For order information see chapter 10 01 2001 gt Mo Bi Tec 5 1 1 Grow n Glow Yeast Growth Media Notes DOB Drop Out Base DOBA Drop Out Base with Agar URA signifies medium lacks uracil HIS signifies medium lacks histidine a and YPD agar rich medium 20 g peptone 10 g yeast extract 20 g glucose pH 6 5 17 g agar per litre YPD broth Pour the entire content of a YPD broth bag order 4001 1 into a 0 5 flask add 500 ml H O Autoclave Cool to at least 37 C Ready for use YPD agar Pour the entire content of a YPD agar bag MoBiTec order 4001 2 into a 0 5 flask add 500 ml H O Autoclave Cool to 55 C Pour into plates Ready for use b YNB URA and or HIS selective medium with glucose DOB glucose 26 7 g l 1 7 g YNB 5 g ammonium sulphate 20 g glucose DOBA DOB glucose with 17 g agar per liter CSM Complete Supplement Mixture The formulation of CSM is a dropout supplement for virtually all strains of S cerevisiae containing different combinations of amino acids adenine and uracil Cells grow vigorously in DOB or DOBA supplemented with CSM DOB URA glucose Pour the entire content of a DOB glucose bag MoBiTec order 4025 1 and 0 385 g CSM URA MoBiTec order 4511 2 into a 0 5 flask add 500 ml H O Autoclave Cool to at least 37 C Ready for use 01 2001 MoBi
23. erile distilled water and spread 100 pl of this dilution onto separate appropriate selective DOBA plates e g HIS URA Incubate at 30 C for 2 3 days until colonies appear Calculate the number of transformants obtained by counting the number of colonies on the plate 200 colonies on the plate corresponds to an efficiency of 2 x10 yg library DNA Thus 100 library DNA will yield 100 x 2 x 104 2 x 10 transformants A saturating screen of e g a mammalian library requires at least 2 x 10 transformants m Streak 4 colonies from each plate onto another DOBA Glu HIS URA plate Incubate at 30 C 2 3 days Perform a GFP assay with a UV lamp see 7 5 01 2001 46 26 MoBi Tec Note For yeast transformation procedures especially electrotransformation electroporation see Ausubel et al 1997 Materials 10 X TE 10 X LiOAc Polyethylene Glycol 1 X TE LIOAc 1 X TE LIOAc PEG Carrier DNA 100 mM Tris HCl pH 7 5 10 mM EDTA 1 M lithium acetate 50 w v Polyethylene Glycol 3350 in H 0 Right before use mix 1 part 10 X TE 1 part 10 X LiOAc 8 ports sterile distilled water Right before use mix 1 part 10 X TE 1 part 10 X LiOAc 8 parts 50 76 PEG 3350 Sonicated salmon or herring sperm DNA Sigma 20 mg ml size 10 kb 01 2001 gt 27 MoBi Tec Appendix Ill Isolation of Plasmids from Yeast a Grow a yeast colony in 2 ml DOBA glu HIS URA overni
24. ession under a UV lamp The colonies that appear positive at this point are further characterized see 7 6 Note Check colonies on plates without lid for fluorescence using a UV handlamp in a darkroom 01 2001 MoBi Tec Caution When using ultraviolet UV radiation it is important to protect yourself with a full length UV blocking face shield and other appropriate skin covering gar ments In order to avoid damage to the DNA it is best to minimize the exposure time of the yeast to the UV light The shortest duration possible is the best Exposing any plate of yeast to UV for more than 2 minutes total does not provide reliable results As much as possible try to maintain sterile technique when working with yeast Disclaimer MoBiTec is not responsible or liable for any harm or damage to any person or to any DNA as a result of exposure to UV radiation 7 6 Further Characterization of Putative Positives Putative positive colonies are picked from the master plate and are inoculated in 3 ml DOB glu HIS URA medium supplemented with CuSO and are grown overnight Freeze 1 ml of every culture in 20 glycerol at 80 C If the number of potential positives is small lt 50 then all should be recovered and further characterized If gt 50 potential positives are obtained then you should charac terize the first 50 that arise and freeze the rest in 1 ml aliquots at 80 C in 20 glycerol 7 6 1 Plasmid Isola
25. ght or until culture shows abundant yeast growth b Spin down 1 5 ml at 14 000 g for 1 minute c Discard supernatant d Resuspend in 200 yl lysis buffer e Add 100 pl of phenol and 100 pl of chloroform isoamylalcohol see below f Add 0 3 g 200 yl glass beads see below g Vortex at maximal speed for at least 5 minutes h Centrifuge at 14 000 g for 10 minutes i Transfer supernatant carefully to fresh tube i Add 8 pl 10 M ammoniumacetate Mix k Add 500 pl 96 ethanol Place at 20 C for at least 30 minutes or 10 minutes at 70 C m Centrifuge at 14 000 g for 10 minutes n Discard supernatant and dry pellet o Resuspend in 20 pl sterile TE buffer Materials Lysis buffer 2 Triton X 100 1 SDS 100 mM NaCl 10 mM Tris HCl pH 8 0 1 mM EDTA TE buffer 10 mM Tris HCl pH 7 5 1 mM EDTA Phenol Tris saturated pH 8 0 01 2001 t 28 MoBi Tec Chloroform Isoamylalcohol 24 1 v v Glass beads acid washed 400 600 nm glass beads Sigma Appendix IV Transformation of competent E coli Cells Competent cells a Inoculate one colony of the E coli strain in 2 5 ml SOB medium see below and incubate overnight at 37 C Note Use a freshly streaked plate with your E coli strain of choice b Incubate 250 ml SOB with 2 5 ml overnight culture Incubate at 37 C and 200 rpm Once an of about 0 4 to 0 5 is reached after about 2 to 2 5 hours place cells on ice
26. h that if Dilution 1 is considered undiluted Dilution 2 1 10 diluted Dilution 3 1 100 diluted and Dilution 4 1 1000 diluted f Plate 100 pl of each of Dilutions 1 4 onto DOBA glu HIS plates and onto DOBA glu HIS plates supplemented with 30 pM CuSO g Incubate at 30 C for 1 3 days You should see colonies on the HIS plates but not on the HIS plates containing CuSO Note If you do obtain many colonies on the HIS plates containing CuSO then your bait is autoactivating and you should perform the assays again using CuSO concentrations higher than 30 pM 30 100 pM If you do not obtain the expected results with a more stringent selection for copper resistance you can try to subclone parts of your bait gene in order to delete those sections responsible for the autoactivation activity If you are failing again to obtain the expected results with this control assay you should not attempt a two hybrid screening with the bait anymore Test Plasmids Growth Autoactivation Test Bait plasmid pTY137 with insert Positive control Control plasmid pTY139 Negative control Bait plasmid without insert Table Expected results of CUP autoactivation by the bait protein cloned into pTY 137 Yeast is grown on DOBA glu HIS plates supplemented with 30 pM 7 3 Autoactivation of GFP A two hybrid system library screen does not have to be performed if the bait protein activates the
27. ions on an agarose gel Clones with an amplified fragment contain a prey plasmid with an insert of interest e Go back to the cultures see point 7 6 3 a Isolate plasmids from the cultures using the Grow n Glow Yeast Plasmid Isolation Kit MoBiTec order 2069 1 which can also be used for bacterial plasmid purification Alternatively use another appropriate method for plasmid purification f Sequence the prey plasmid insert with the 5 PREYprimer Use the DNA sequence to search against a database of choice Performing a PCR with the 5 PREYprimer and 3 PREYprimer using the prey plasmid carrying no insert as template results in a 237 bp DNA fragment 01 2001 MoBi Tec 7 7 Other Verification Methods of Protein Protein Interaction The protein protein interaction should always be confirmed by an independent method One such method is purifying your bait and prey proteins e g by means of our PheBo or pAX protein expression and purification systems and co immunoprecipitation analysis using specific antibodies against your bait and prey proteins Moreover you can also verify your results by switching cloning vectors i e moving the library insert from the AD to the DNA BD vector and vice versa and then repeat the two hybrid assay or using a different two hybrid system e g MoBiTec s LexA based Grow n Glow GFP Two Hybrid System GNGKO1 Another possibility is generating mutations of your prey by deletions su
28. l B D galactopyranoside X Gal This latter step can often be time consuming depending on the number of primary transformants obtained In order to simplify and accelerate the screening process of the two hybrid systems and improve selection of positive clones a yeast strain was constructed containing both the gene encoding the green fluorescent protein GFP from the bioluminescent cnidarian Aequorea victoria and the metallothionein encoding gene 1 2 Background MoBiTec s based Grow n Glow yeast two hybrid system is a version of a 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 technology is based onthe factthat many eukaryotic transcriptional activators such as GALA consist of two physically separable modular domains one DNA binding domain and one transcriptional activation domain In order to activate transcription they have to be physically tethered to one another and neither of them can activate transcription on its own The same basic idea is followed in ACE1 based yeast two hybrid systems where the DNA binding and transcriptional activation domains of the yeast transcription factor ACE1 are used Binding of this transcription factor to DNA is dependent on the presence of copper ions that induce a conformational change in the N terminus of AC
29. litre medium each 4001 1 4001 6 4001 2 4001 7 4025 1 4025 6 4026 1 4026 6 3002 1 3002 6 3002 2 3002 7 Powder 4510 3 4511 2 4520 3 YPD broth bags with 25 g for 0 5 litre medium each 10 bags YPD broth bags 10 x 10 bags agar bags with 33 5 g for 0 5 litre medium each 10 bags YPD agar bags 10 x 10 bags DOB glucose bags with 13 4 g for 0 5 litre medium each 10 bags DOB glucose bags 10 x 10 bags DOBA glucose bags with 21 9 g for 0 5 litre medium each 10 bags DOBA glucose bags 10 x 10 bags LB medium bags with 12 5 g for 0 5 litre medium each 10 bags LB medium bags 10 x 10 bags LB agar bags with 20 g for 0 5 litre medium each 10 bags LB agar bags 10 x 10 bags CSM HIS supplement 10g CSM URA supplement 10g CSM HIS URA supplement 10g DOB Drop Out Base DOBA Drop Out Base with Agar t 32 01 2001
30. n of copper should give clearly reduced GFP activities In non inducible systems the reporter gene e g B galactosidase is expressed during the whole fermentation process of the yeast cells before the addition of the inhibitory compound Thus the already existing level of the reporter protein makes it difficult to measure any inhibitory effect 01 2001 gt Mo Bi Tec 1 3 GFP The GFP of the jellyfish A victoria is activated in vivo by an energy transfer via the Ca stimulation of the photoprotein aequorin The blue light generated by aequorin excites GFP and results in the emission of green light GFP itself consists of 238 amino acids Mr 27 kDa and is synthesized as an apoprotein in which post translational formation of the chromophore occurs in an O dependent manner independent of any other gene products 4 It maximally absorbs light at 395 nm and has an emission peak of 509 nm The nonsubstrate requirement for GFP activity makes this protein an attractive reporter for gene expression studies and this utility was initially demonstrated in both prokaryotes Escherichia coli and eukaryotes Caenorhabditis elegans It has subsequently been used to monitor gene expression in many organisms including mouse Drosophila zebrafish embryos Arabidopsis and yeast 4 In addition to the non invasiveness of GFP detection long wave UV light the protein is very stable non toxic and resistant to photobleaching The
31. of the GFP re porter gene itself and enables its host to grow on copper containing me dium It contains the entire Acel cod ing sequence DNA binding and ac tivation domain under control of the Acel promoter and a HIS3 marker AmpP Ace1p ACE1 ARsu PTY139 ori CYCit Hind Il Hind 11 Pstl 4 3 Primer 5 BAlTprimer 5 GGG AGG 3 5 forward primer for sequencing the junction between ACE1BD and the gene of pTY137 5 PREYprimer 5 ACT ACG AAC ACA GCA C 3 5 forward primer used to determine the reading frame and identity of positive clones in pTM114 can be used with 3 PREYprimer to amplify clone by PCR 3 PREYprimer 5 GAC CTA GAC TTC AGG T 3 3 primer used to determine the identity of positive clones in pTM1 14 can also be used with 5 PREYprimer to amplify a clone by Polymerase chain reaction PCR is a patented process owned by Hoffmann LaRoche AG 01 2001 MoBi Tec 5 Materials Required but not Supplied Note The specific materials listed below are the ones we have tested in the Grow n Glow System For order information see chapter 10 Similar items from other sources may be interchangeable 1 Grow n Glow Yeast Growth Media see chapter 5 1 1 2 Grow n Glow Bacterial Growth Media see chapter 5 1 2 3 Ampicillin Roche Molecular Biochemicals order 835269 4 For yeast transformations Grow
32. r resistance mediating CUP1 reporter gene also stably integrated in the yeast chromosome allows a more stringent and even modula tory selection of positive clones on media containing copper ions Discrimination between strong or weak protein interactions at an early step can be achieved by different copper concentrations in the medium Since only those cells expressing well interacting proteins can endure the toxicity of copper ions the stringency of the CUP1 mediated growth selection can be modulated by different concentrations of CuSO Strong interactors should be distinguished readily from weak interactors by comparing CUP1 expression the growth of yeast cells expressing low affinity interacting proteins will be much more depressed on copper containing medium than that of cells expressing proteins with a high binding affinity to one another Furthermore weak interactors determine weak GFP expression the GFP signal of those cells is greatly reduced compared to cells expressing strongly interacting hybrid proteins Furthermore this novel two hybrid system is also suited for the development of high throughput screening assays to screen for compounds affecting the inter action between therapeutic relevant proteins Since the GFP reporter expression is initiated solely by the addition of copper ions binding of the ACE1BD to DNA is dependent on the presence of copper ions incubation of cells with an inhibitory drug followed by the additio
33. reporter genes on its own To test for autoactivation by your bait fusion protein transform yeast strain ITH5 with the following combinations of vectors 01 2001 t MoBi Tec Plasmids Plates Expected Results pTY137 with insert DOBA glu HIS No Fluorescence Test for GFP autoactivation pTY137 without insert DOBA glu HIS No Fluorescence Negative control pTY137 with insert pTM114 without insert DOBA glu URA HIS No Fluorescence Test for GFP autoactivation pTY139 DOBA glu HIS Fluorescence Positive control pTY143 pTM125 DOBA glu URA HIS Fluorescence Positive control Table Expected results of testing for GFP autoactivation by the bait protein cloned into pTY137 Expected results The colonies containing pTY139 or pTY143 pTM125 should emit green light the colonies containing bait plasmid pTY137 without insert or pTY137 pTM1 14 without insert should not fluoresce green and the colonies containing bait plasmid pTY137 with insert may or may not emit green light If yeast colonies with your bait protein do not fluoresce green then the bait does not autoactivate reporter gene expression and can be used for screening If the clones containing your bait protein or the bait protein and the empty prey vector 1 14 do emit green light in the above assay then you must subclone only parts of the gene encoding your protein into bait plasmid pTY13
34. rs at 37 C Note For E coli transformation procedures especially electrotransformation electroporation see Ausubel et al 1997 or Sambrook et al 1989 5 Protocols for the isolation of plasmid DNA from E coli can also be found in both manuals 01 2001 MoBi Tec 10 Order Information Shipping amp Storage order 4 ACEO description amount Grow n Glow ACE1 Two Hybrid System Complete Kit pTY137 lyophilized DNA 114 lyophilized DNA pTY143 lyophilized DNA pTM125 lyophilized DNA pTY139 lyophilized DNA 5 BAlTprimer 5 PREYprimer 3 PREYprimer Yeast strain ITH5 glycerol stock shipped on dry ice vectors amp primers stored at 4 C yeast strains stored at 20 C Related MoBilec Products order GNGKO3 GNGKO1 GNGK02 2100 1 2200 1 2069 1 2069 2 STAQO2 description Grow n Glow One Hybrid System Grow n Glow Two Hybrid System Complete Kit Grow n Glow Two Hybrid System Basic Kit Grow n Glow Fast and Easy Yeast Transformation Kit Grow n Glow High Efficiency Yeast Transformation Kit Grow n Glow Yeast Plasmid Isolation Kit Grow n Glow Yeast Plasmid Isolation Kit Supertaq thermostable polymerase MoBiTec 500 pmole 1 ml amount Kit Kit Kit 200 transf 250 transf 25 preps 100 preps 250 units 01 2001 gt 31 Mo Bi Tec Grow n Glow Yeast and Bacterial Growth Media order description amount Bags for 0 5
35. se properties make GFP a viable alternative to traditional reporter genes such as B galactosidase LacZ B glucuronidase GUS chloramphenicol acetyl transferase CAT or firefly luciferase which require substrate for their detection Several modifications of the wild type GFP cDNA have been engineered with optimized codon usage improved fluorescence activity and red shifted variants with altered excitation maxima intended for fluorescence microscopy The A victoria GFP variant GFPuv is optimized for maximal fluorescence by UV light excitation making it fluoresce 18 times brighter than wildtype GFP while retaining identical excitation and emission wavelength maxima The GFP reporter allows a very sensitive direct detection in living cells without the time consuming necessity of preparing cell extracts Clones expressing interact ing proteins are easily detected by placing the plate with the colonies under a UV handlamp in the dark room Since the detection of the GFP signal does not require cell lysis it allows the monitoring of reporter gene activity as a function of time especially if a fluorescence spectrophotometer is used for measurement of whole cell fluorescence Legend to flow chart on page 7 Identification of molecular interactions with the Grow n Glow ACE1 Two Hybrid System To test whether the bait protein activates the reporter genes on its own autoactivation only the bait vector with the target gene istransformed into yeast
36. tion from Yeast To isolate DNA from the potential positives we recommend the Grow n Glow Yeast Plasmid Isolation Kit to obtain best results MoBiTec order 2069 1 Alternatively you can use the procedure in Appendix III 7 6 2 Transformation of E coli with Plasmids Isolated from Yeast Use standard transformation procedure CaCl or electroporation procedures Sambrook et al 198915 Ausubel et al 1997 5 or the protocol listed in Appendix IV Use 5 pl plasmid DNA for Grow n Glow Yeast Plasmid Isolation Kit users to transform E coli spread the transformation solution onto LB Amp agar plates and incubate at 37 C overnight Colonies arising at this stage contain either the bait or the prey plasmid 01 2001 gt 21 MoBi Tec 22 7 6 3 Prey Plasmid Identification by PCR Follow the protocol a to f a Inoculate 6 colonies from each plate in 2 ml LB Amp medium Grow overnight at 37 C b 5 pl culture are transferred to a PCR tube and centrifuged for 5 sec at maxima speed Discard supernatant Add 30 pl PCR mix and mix PCR mix 20 pmole 3 PREYprimer 20 pmole 5 PREYprimer 3 yl 10 X dNTP 2 5 mM dNTP 3 pl 10 X Taq DNA polymerase buffer Taq DNA polymerase use appropriate amount as suggested by supplier H O up to 30 pl c Run the reaction in a thermal cycler as follows 1 cycle 2 min 95 C 20 cycles 30 sec 95 C 30 sec 60 C 120 sec 72 C 1 cycle 5 min 72 C d Load the PCR react
37. two hybrid system was developed by Dr Thomas Munder at the Hans Knoll Institut Jena Germany General knowledge Users of the kit should be familiar with basic molecular biology and microbiological techniques NOTE FOR RESEARCH PURPOSES ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USAGE We will replace at no cost any product of ours that does not meet our standard product specifications No other warranties expressed or implied are given with our products MoBiTec GmbH is not liable for any damages due to the use of this product nor are we liable for the inability to use this product PLEASE NOTE THAT THIS KIT I FOR USE BY THE PURCHASER ONLY AND IS NOT TO BE DISTRIBUTED TO THIRD PARTIES WITHOUT THE WRITTEN CONSENT OF MOBITEC GMBH Licence for GFP This product is sold under licence from Columbia University USA Rights to use this product are limited to research use only NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rights are conveyed Inquiry into availability of a license to broader rights or the use of this product for commercial purposes should be directed to Columbia Innovation Enterprise Colombia University Engineering Terrace Suite 363 New York New York 10027 USA Licence for Two Hybrid Technology Practice ofthe two hybrid system is covered by U S Patents Nos 5 283 173 5 468 614 and 5 667 973 assigned to The Research Foundation of the State University of New York USA Purchase of any two h
38. ybrid reagents does not imply or convey a licence to practice the two hybrid system covered by these patents in the U S A practicing U S Patent No 5 283 173 must obtain a licence from The Research Foundation of State University of New York Non profit institutions may obtain a complimentary licence for research not sponsored by industry Please contact Barbara A Sawitsky at The Research Foundation of SUNY at Stony Brook lor licence information phone 516 632 4163 lax 516 632 9839 01 2001 P MoBi Tec 1 Introduction 1 1 General The yeast two hybrid system or interaction trap has rapidly become a widely used technique in molecular biology It is now the method of choice to identify protein protein interactions from either cDNA libraries or known gene se quences The method relies on the transactivation of reporter genes in Saccharomyces cerevisiae to identify positive interactions In common two hybrid systems growth selection via activation of either a HIS3 or LEU2 reporter gene is used in conjunction with a second reporter gene such as LacZ which expresses B galactosidase The use of two reporter genes enables discrimina tion of false positives which activates only the auxotrophic marker Yeast colonies that survive the growth selection scheme are chosen re streaked and tested for expression of the LacZ gene by means of a filter assay or growth on minimal medium plates containing 5 bromo 4 chloro 3 indoly
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