Grow`n`Glow: The GFP One
Contents
1. 27 Tips for a Successful Transformation 27 Small Scale Yeast Transformation Protocol 28 Appendix II Competent E coli Cells and Transformation 29 Competent Cells 29 Bulla aaa 30 Transformation of Recombinant Plasmids into E coli 30 Appendix Ill Order Information 31 Related MoBiTec Products 31 The Grow n Glow system was developed by Dr Robert S Cormack and Dr mre E Somssich at the Max Planck Institut f r Z chtungsforschung Cologne Germany The system is patented 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 the use of this product nor are we liable for the inability to use this product PLEASE NOTE THAT THIS KIT IS 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 Rights to use this product are limited to research use only NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rahi are conveyed2. 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 Columbia University Engineering Terrace Suite 363 New York New York 10027 USA pGNG2 12 2002 gt 3 MoBi Tec 1 Introduction 1 1 General The Grow n Glow GFP One Hybrid Kit isolates genes for proteins that bind a specific DNA element of interest Strubin ef al 1995 and Luo etal 1996 In addition to finding novel DNA binding proteins the one hybrid system can be used to investigate the bases and amino acids involved in specific DNA protein interactions Proteins can be found that bind to any short DNA element of interest The Grow n Glow system offers maximal sensitivity because detection of the DNA protein interaction occurs in vivo where proteins are more likely to be in their native conformation Use of the Green Fluorescent Protein GFP reporter eliminates the time consuming B galactosidase lift assays The particular GFP variant expressed by the GFP reporter plasmid GFPuv has the same excitation and emission maxima as wild type GFP but is 18 times brighter than the wild type variant Crameri etal 1996 lt is easily detected by placing the plate without the lid with the yeast colonies on a standard 300 nm UV transilluminator or better under a UV hand lamp in a darkroom With this sensitive variant positive colonies with DNA binding proteins glow a
3. Grow n Glow High Efficiency Yeast Transformation kit MoBiTec order 2200 1 or the Grow n Glow Fast and Easy Yeast Transformation kit MoBiTec order 2100 1 which are optimized for two and one hybrid systems The pGNG2 12 2002 Mo Bi Tec Grow n Glow Yeast Transformation kits contain all the solutions required for transformation Use the protocol supplied with the Grow n Glow Yeast Transformation kits using 200 ng of plasmid DNA for transformation or the procedure described in Appendix l 7 3 Screening a Grow n Glow cDNA Library for DNA Binding Protein Genes 7 3 1 Reagents and Materials Required e DOB URA and DOB TRP URA Glu medium chapter 5 1 1 e DOB TRP URA Gal Raf agar plates medium chapter 5 1 1 e 15 sterile 245 x 245 x 25 mm Nunc Bio Assay Dishes Nunc 240835A e Appropriate sterile tubes and flasks Notes a Prepare the selection media and pour the required number of agar plates in advance You will need 15 Petri dishes 22 x 22 cm DOB TRP URA at 37 C for the transformation b Allow DOB agar plates to dry unsleeved at room temperature for 2 3 days or at 30 C for 3 hours prior to plating transformation mixtures EGY48 yeast strain e Grow n Glow library plasmid DNA in solution Your prey library should have at least 105 clones For information on constructing your own libraries see Vojtek 1993 Durfee et al 1993 and Dalton and Treisman 1992 Herring testes carrier DNA Sig
4. Incubate plate at 30 C for 1 3 days until colonies appear Seal this working stock plate with Parafilm and store at 4 C Propagate additional cultures only from isolated single colonies on this plate You should restreak this plate every 2 3 weeks Note The cells may have settled to the bottom of the tube before the stock was frozen If this happens thaw the frozen culture on ice and vortex it before restreaking The stock can be refrozen Healthy yeast colonies grow to gt 2 mm in diameter Avoid small white colonies lt 1 mm which are cells with spontaneous mutations 1 2 that eliminate mitochondrial function Holm 1993 when you inoculate a culture 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 genetic and molecular biological methods due to its ability to be transformed by foreign DNA and its highly efficient system of homologous recombination General knowledge Users should be familiar with basic molecular biology and microbiological techniques Ausubel et al 1997 Sambrook ef al 1989 7 Grow n Glow One Hybrid System Protocol 7 1 Reporter Bait Constructs 7 1 1 GFP Reporter Vector
5. glucose bags 10 x 10 bags 4025 2 2 galactose 1 raffinose powder 0 5 b 227 g 4025 7 DOB 2 galactose 1 raffinose powder 2 2 lb 1 kg 4026 2 DOBA 2 galactose 1 26 raffinose powder 0 5 b 227 g 4026 7 DOBA 2 galactose 1 raffinose powder 2 2 lb 1 kg 4026 1 DOBA glucose bags with 21 9 g for 0 5 litre medium each 10 bags 4026 6 DOBA glucose bags 10 x 10 bags 3002 1 LB medium bags with 12 5 g for 0 5 litre medium each 10 bags 3002 6 LB medium bags 10 x 10 bags 3002 2 LB agar bags with 20 g for 0 5 litre medium each 10 bags 3002 7 18 agar bags 10 x 10 bags 4510 3 CSM HIS supplement 10g 4511 2 CSM URA supplement 10g 4520 4 CSM HIS LEU supplement 10g 4520 3 9 HIS URA supplement 10g 4520 5 CSM TRP URA supplement 10g 4530 8 CM HIS TRP URA supplement 10g 4540 0 CSM HIS LEU TRP URA supplement 10g DOB Drop Out Base DOBA Drop Out Base with Agar pGNG2 12 2002 32
6. the bait element 1 The hybrid protein cannot be localized to the yeast nucleus see van Aelst et al 1993 for one example In these cases it may help to construct hybrids containing different domains of the DNA binding protein For example to study proteins that normally do not localize to the nucleus it may be necessary to generate mutant forms of the protein that can be transported across the nuclear membrane 2 The hybrid protein folds improperly 3 The B42 activator occludes the site of interaction 4 The hybrid proteins are not stably expressed in the host cell Solution 2 If according to the control in chapter 7 3 4 the transformation efficiency is too low you may not be screening a sufficient number of library co transformants This can be critical especially if a rare transcript in the source tissue encodes the interacting protein of interest See chapter 7 5 2 above for tips on improving transformation efficiency pGNG2 12 2002 Mo Bi Tec 8 Literature Arndt K T Styles C und Fink G R 1987 Multiple global regulators control HIS4 transcription in yeast Science 237 874 880 Ausubel F M Brent R Kingston R E Moore D D Seidmann J G Smith J A and Struhl K 1997 Current Protocols in Molecular Biology John Wiley and Sons Inc New York Challie M Tu Y Euskirchen G Ward W W and Prasher D C 1994 Green fluorescent protein as a marker for gene expression Sc
7. use DOBA TRP URA galactose raffinose Pour 18 4 g DOBA Gal Raf MoBiTec order 4026 2 and 0 36 g CSM TRP URA order 4520 5 into a 0 5 flask add 500 ml H O Autoclave Pour into plates Ready for use 5 1 2 Grow n Glow Bacterial Growth Media a LB Medium Pour the entire content of a LB medium bag MoBiTec order 3002 1 into a0 51 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 a LB agar medium bag MoBiTec order 3002 2 into a 0 5 flask add 500 ml H O Autoclave Cool to at least 55 C Pour into plates c LB Amp Medium ampicillin selection Cool the LB Medium above to at least 55 C and add 2 ml of 50 mg ml ampicillin in distilled water filter sterilized per litre of medium Mix d LB Amp Agar Medium ampicillin selection Cool the LB Agar Medium above to 55 C and add 2 ml of 50 mg ml ampicillin in distilled water filter sterilized per litre of medium Mix Pour into plates pGNG2 12 2002 gt 13 Mo Bi Tec 14 6 Growth and Maintenance of Yeast The yeast strain Saccharomyces cerevisiae see chapter 4 3 in our kit is provided in YPD medium with 15 glycerol and can be maintained indefinitely at 70 C To recover the yeast strain from the frozen glycerol stock scrape a small amount of frozen cells from the frozen stock with a sterile loop or wooden stick and streak them onto a YPD plate
8. use YPD Agar Pour the entire content of the YPD agar bag MoBiTec order 4001 2 into a 0 51 flask add 500 ml H O Autoclave Pour into plates Ready for use b DOB media and DOBA agar with glucose or galactose raffinose selective medium 1 7 g yeast nitrogen base 5 g ammonium sulfate 20 g glucose or 20 g galactose and 10 g raffinose 17 g agar per litre DOB URA glucose Pour the entire content of a DOB glucose bag MoBiTec order 4025 1 and 0 385 g CSM URA order 4511 2 into a 0 51 flask add 500 ml H O Autoclave Ready for use DOBA URA glucose Pour the entire content of a DOBA glucose bag MoBiTec order 4026 1 and 0 385 g CSM URA MoBiTec order 4511 2 into a 0 5 flask add 500 ml H O Autoclave Pour into plates Ready for use DOB TRP URA glucose Pour the entire content of a DOB glucose bag MoBiTec order 4025 1 and 0 36 g CSM TRP URA MoBiTec order 4520 5 into a 0 5 flask add 500 ml H O Autoclave Ready for use pGNG2 12 2002 Mo Bi Tec DOBA TRP URA glucose Pour the entire content of a DOBA glucose bag MoBiTec order 4026 1 and 0 36 g CSM TRP URA MoBiTec order 4520 5 into a 0 51 flask add 500 ml H O Autoclave Pour into plates Ready for use DOB TRP URA galactose raffinose Pour 18 4 g DOB Gal Raf MoBiTec order 4025 2 and 0 36 g CSM TRP URA MoBiTec order 4520 5 into a 0 5 flask add 500 ml H O Autoclave Ready for
9. 1 Plate 1ml of the transformation mixture on each 245 x 245 mm plate 15 plates total containing DOB TRP URA Gal Raf agar chapter 5 1 1 which stimulate the expression of the fusion proteins of the prey plasmid Spread the cells immediately after pipetting them onto the plate 2 Incubate at 30 C for 2 4 days 3 Place the plates without the lid under an UV light 365 nm in a darkroom Localize and isolate all colonies emitting green light using a wooden stick and streak onto a new fresh 100 mm DOB TRP URA Gal Raf plate A Incubate the isolated and restreaked putative positives at 30 C for 2 4 days 5 Place the plates without the lid under an UV hand lamp in a darkroom Identify the colonies emitting green light and inoculate these in 5 ml DOB TRP URA Glu medium Incubate at 30 C for 3 hours with shaking at 230 rpm Continue with yeast plasmid purification see chapter 7 3 4 7 3 4 Isolating the Prey Plasmid from Putative Positive Yeast Colonies We recommend the Grow n Glow Yeast Plasmid Isolation kit MoBiTec order 2069 1 providing the reagents for a fast and simple protocol for isolating plasmids from yeast This procedure will provide plasmid DNA suitable for PCR and E coli transformations Otherwise you can use the methods described by Ausubel ef al 1997 We recommend that the identity of the selected clones is confirmed by indepen dent methods First E coli cells are transformed with plasmids i
10. 7 onto a LB 50 pg ml Amp plate Incubate overnight at 37 C pGNG2 12 2002 Appendix III Order Information Shipping amp Storage Mo Bi Tec order description amount GNGK03 Grow n Glow One Hybrid System pGNG2 lyophilized DNA 5 pg p G4 5 lyophilized DNA 5 pg GNGprimer 500 pmole 5 PREYprimer 0 1 nmole pl 500 pmole 3 PREYprimer 0 1nmole pl 500 pmole Yeast strain EGY48 glycerol stock 1 ml shipped on dry ice vectors and primers store at 4 C yeast strain store at 20 C Related MoBiTec Products order 2 description amount GNGKO1 Grow n Glow Two Hybrid System complete kit GNGKO2 Grow n Glow Two Hybrid System basic kit kit 1 Grow n Glow ACE1 Two Hybrid System complete kit kit 2100 1 Grow n Glow Fast and Easy Yeast Transformation kit 200 transf 2200 1 Grow n Glow High Efficiency Yeast Transformation kit 250 transf 2069 1 Grow n Glow Yeast Plasmid Isolation kit 25 preps 2069 2 Grow n Glow Yeast Plasmid Isolation kit 100 preps pGNG2 12 2002 gt MoBi Tec Grow n Glow Yeast and Bacterial Growth Media order description amount Bags for 0 5 litre medium each 4001 1 YPD broth bags with 25 g for 0 5 litre medium each 10 bags 4001 6 broth bags 10 x 10 bags order 2 description amount 4001 2 YPD agar bags with 33 5 g for 0 5 litre medium each 10 bags 4001 7 agar bags 10 x 10 bags 4025 1 DOB glucose bags with 13 4 g for 0 5 litre medium each 10 bags 4025 6
11. 9 MoBi Tec 30 Buffers SOB 500 ml 2 w v Bacto Tryptone or peptone 0 5 w v yeast extract 10 mM NaCl 2 5 mM 10 mM MgC 10 mM MgSO Autoclave RF buffer 400 ml 100 mM RbCl rubidium chloride 30 mM KAc 10 mM CaCl 50 mM 15 Glycerol Adjust to pH 5 8 with 0 2 M acetic acid Autoclave RF II buffer 100 ml 10 mM MOPS 10 mM RbCl 75 mM 15 glycerol distilled H O to approximately 75 ml Adjust to pH 6 8 with 1 M NaOH Add H O up to 100 ml Autoclave Transformation of Recombinant Plasmids into E coli Note on controls In addition to the experimental transformations described below we recommend that you perform a negative control transformation with no DNA and a positive control transformation with 0 1 ng of an intact plasmid such as pBR322 or pUC19 0 1 ng pUC19 should result in 100 1000 colonies 1 Thaw the competent E coli cells on ice to obtain maximum efficiency Caution Once thawed cells cannot be refrozen 2 Place 5 pl plasmid for Grow n Glow Yeast Plasmid Isolation kit users on ice in an 1 5 ml Eppendorf tube Add 220 pl competent cells immediately when the cells are thawed Incubate on ice for 30 minutes Incubate at 42 C for exactly 2 minutes Place immediately on ice for 15 minutes Add 200 pl LB medium Incubate by shaking at 37 C for 1 hour Place on ice for 5 minutes Oo ON A Oa BR W Plate the whole volume from step
12. Mo Bi Tec Grow n Glow The GFP One Hybrid System Product Information and Instructions December 2002 nin msn d JOLECULAR BIOLOGISCHE TECHNOLOGIE MoBi Tec Content page 1 inirodushen ua basan 24 1 1 General a 4 1 2 Bek GrOUNG auscrsisacdienctcnas 5 1 3 Green Fluorescent Protein GFP 5 2 Advantages of the Grow n Glow System 6 3 Schematic Overview of the Grow n Glow System 7 4 Kit Components n nal m B 4 1 DNA ae N 8 4 1 1 GFP Reporter Vector pGNG2 2222 8 4 1 2 Prey Plasmid pJG4 5 9 ANS Control Plasm dsa ab aba 9 4 2 rmeruo DB 10 4 2 1 GING PRINS irda aaa A A RD M 10 42 25 OAPREV MICH seek 10 AZO 0 ERET T m T Da 10 4 3 Yeast OU d lar 10 5 Materials Required but not Supplied 11 5 1 Recipes for Media V 11 5 1 1 Grow n Glow Yeast Growth Media 12 5 1 2 Grow n Glow Bacterial Growth Media 13 6 Growth and Maintenance of Yeasf 14 7 Grow n Glow One Hybrid System Protocol 14 7 1 Reporter Bait 14 7 1 1 GFP Reporter and Prey Plasmid 14 7 1 2 Element Tandem Cop
13. ait plasmid 7 5 2 F Transformation Efficiency When Screening a Grow n Glow Library The transformation efficiency should be at least 104 cfu pg for the library transformation If your library transformation efficiency is lower than this try one or more of the following suggestions Solution 1 Repeat the experiment using more of the prey library plasmid Check the purity of the DNA and if necessary re purify it by ethanol precipitation before using it again If you are not already doing so we strongly recommend using the pre tested and optimized carrier DNA which is available as part of the Grow n Glow Yeast Transformation kits MoBiTec order 2100 1or 2200 1 Solution 2 Repeat the transformation this time including a recovery period after the heat shock To provide a recovery period perform the transformation as described but add the following steps after step 20 1 Resuspend cells in 50 ml of DOB TRP URA Glu medium Divide cell suspension into two 50 ml tubes 2 Incubate cells at 30 C for 1 h with shaking at 230 rpm 3 Centrifuge at 2000x g for 5 minutes at room temperature Remove super natant pGNG2 12 2002 gt 23 MoBi Tec 24 7 5 3 False Negative Results A protein that normally interacts in vivo does not interact with the bait element Solution 1 If one of the following situations is occurring it may interfere with the ability of the B42 activator hybrid proteins to interact with
14. and Prey Plasmid In order to screen a cDNA for DNA binding proteins using the Grow n Glow One Hybrid System a true or putative bait element has to be identified The bait element must be precisely defined using for example deletion and or point mutation analysis Prepare a construct composed of three or more tandem copies of your bait regulatory element bordered by restriction sites and insert it upstream of the reporter gene in the multiple cloning site of the GFP reporter pGNG2 12 2002 Mo Bi Tec plasmid This may alter the level of background GFPuv expression Therefore constructs should always be tested for GFPuv expression under UV light The prey plasmid carries the GAL promoter which is induced by galactose and inhibited by glucose This feature ensures that even toxic proteins can be expressed 7 1 2 Bait Element Tandem Copy Synthesis At least three tandem copies of the bait element should be inserted upstream of the reporter gene Ghosh et al 1993 Various methods e g Liaw 1994 are available to generate tandem copies however we have found the most convenient and reliable method for generating them to be oligonucleotide synthesis since well defined regulatory elements are usually smaller than 20 bp 1 Two antiparallel oligonucleotides are designed one representing the sense strand and the other its antisense complement The sense strand should consist of at least three tandem copies of the bait element with a dif
15. are from 10x TE buffer 100 mM Tris pH 7 5 10 mM EDTA e Sterile glass rod bent pasteur pipette or 5 mm glass beads for spreading transformation mixtures on plates Tips for a Successful Transformation 1 Fresh 1 3 days old colonies will give best results for liquid culture inoculation A single large 2 3 mm colony is used for the inoculum Scrape the entire colony into the medium If colonies on the stock plate are smaller than 2 mm scrape several colonies into the medium see also chapter 71 2 If the overnight or 3 hours cultures are visibly clumped disperse the clumps with vigorous vortexing before using them in the next step 3 When you are collecting cells by centrifugation a swinging bucket rotor results in better recovery of the cell pellet A For the highest transformation efficiency as is necessary for library screen ing use competent cells within 1 hour of their preparation If necessary competent cells can be stored after step 10 at room temperature for several hours with a minor reduction in competence 5 To obtain an even growth of colonies after plating continue to spread the pGNG2 12 2002 gt 27 MoBi Tec 28 transformation mixtures over the agar surface until all liquid has been absorbed Small Scale Yeast Transformation Protocol This protocol is for small scale transformation obtaining 100 1000 transformants 1 Inoculate several colonies of the yeast strain 2 3 mm
16. arget plasmid from positive clones with an insert using an available plasmid purification method Sequence the target plasmid insert with the 5 PREYprimer 7 Use the DNA sequence to search against a database of choice PCR is a process covered by patents owned by Hoffmann La Roche Use of this process requires a licence pGNG2 12 2002 Mo Bi Tec 7 4 Confirmation of DNA Binding Activity Although none of the tests suggested below is independently conclusive the results should provide enough convincing evidence together to support whether the Grow n Glow library plasmid encodes a DNA binding protein 1 You may want fo perform a positive control experiment if a non binding mutant of your target element is available First prepare a mutant type construct in the GFP reporter plasmid otherwise identical to your original target reporter construct Then transform the construct into EGY48 and transform the new reporter strain with the candidate prey library plasmid Fluorescing colonies should result from transcriptional activation using the wild type but not mutant target indicating that you have identified a sequence specific DNA binding protein see Li and Herskowitz 1993 for an example 2 Sequence the positive library clones with the 5 PREYprimer and compare the sequence with that of other DNA binding proteins in GenBank EMBL or other databases a In case your sequencing results reveal a peptide below ten amino ac
17. bright green which is easily seen by eye under UV illumination Expression of fusion proteins by the prey plasmid is controlled by the GAL1 upstream activation sequence In yeast with an intact galactose regulatory system the GAL activation sequence is induced by galactose and repressed by glucose This regulation provides a mean to prevent expression of library activation domain fusions until the actual screening which avoids any poten tially toxic library proteins affecting growth of the yeast These sort of toxic proteins would be missed during screening in a non inducible system For the Grow n Glow One Hybrid assay prepare a yeast reporter plasmid containing the sequence of a specific bait element upstream of the reporter gene GFP For this purpose at least three tandem copies of a known bait element are required The bait elements are then inserted upstream of the reporter gene promoter of the GFP reporter vector using the available unique restriction enzymes chapter 4 1 1 Then this construct reporter bait vector is trans formed into yeast cells and the transformants are selected by their marker gene To screen a library for a gene encoding a DNA binding protein of interest the user transforms the reporter bait strain with a Grow n Glow library of fusions between the bait independent activation domain and potentially bait specific DNA binding proteins The transformed yeast cells are then plated on selective medium plates If a G
18. er enough overnight culture to produce an 0 2 0 3 into 300 ml of DOB URA Glu medium 6 Incubate at 30 C for 3 h with shaking at 230 rpm The OD 0 4 0 5 7 Centrifuge the culture in 50 ml tubes at 2000x g for 5 minutes at room temperature go Will be 8 Discard the supernatant and vortex to resuspend each cell pellet in 25 ml of TE buffer 9 Pool the cells into one tube 10 Centrifuge the cells again at 2000x g for 5 minutes at room temperature 11 Discard the supernatant and resuspend the cell pellet in 1 5 ml of freshly prepared sterile 1x TE LiAc Mix well by vortexing 12 In a sterile 50 ml tube add 20 40 vg of prey plasmid library and 5 mg of carrier DNA and mix well 13 Add 1 ml of competent cells to the step 12 DNA mixture and mix well by vortexing Note If cells are not mixed well transformation efficiency may decline 14 Add 6 ml of sterile 1x PEG LiAc to the transformation mixture 15 Mix well by inverting at least ten times 16 Incubate at 30 C for 30 minutes with shaking at 200 rpm pGNG2 12 2002 Mo Bi Tec 17 Add 700 pl of DMSO and mix well by gentle inversion Do not vortex 18 Heat shock for 15 minutes in a 42 C water bath Occasionally invert carefully to mix 19 Chill on ice for 2 minutes 20 Centrifuge at 2000x g for 5 minutes at room temperature and remove supernatant 21 Resuspend cells in 25 ml of TE buffer 7 3 3 Plating the Transformation Mixture
19. er species In Methods A Companion to Methods in Enzymology 5 102 109 Li J J and Herskowitz 1 1993 Isolation of ORC6 a component of the yeast origin of recognition complex by a one hybrid system Science 262 1870 1873 pGNG2 12 2002 gt 25 MoBi Tec 26 Liaw G J 1994 Improved protocol for directional multimerization of a DNA fragment Biotechniques 17 668 670 Luo Y Vijaychander S Stile J and Zhu L 1996 Cloning and analysis of DNA binding proteins by yeast one hybrid and two hybrid systems Biotechniques 20 564 568 Meng A Tang H Ong B A Farrell M J and Lin S 1997 Promoter analysis in living zebrafish embryos identifies a cis acting motif required for neuronal expression of GATA 2 Proc Natl Acad Sci USA 94 6267 6272 Niedenthal R K Riles L Johnston M and Hegemann J H 1990 Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast Yeast 12 773 786 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratories Cold Spring Harbor NY Strubin M Newell J W and Matthias P 1995 OBF 1 a novel B cell specific coactivator that stimulates immunoglobin promoter activity through association with octamer binding proteins Cell 80 497 506 van Aelst L Barr M Marcus S Polverino A and Wigler M 1993 Complex formatio
20. ferent restriction site on each end When the two strands are annealed the resulting double stranded DNA will have a different overhang at each end for directional cloning into the reporter plasmid s multiple cloning site 2 Synthesize both strands without 5 phosphates 7 1 3 Tandem Copies of Bait inserted Upstream of GFP Reporter Gene 7 1 3 1 Reagents and Materials Required e Bait element Sense and antisense strand oligonucleotide e Competent E coli cells Sambrook et al 1989 e T4 DNA ligase 10x T4 ligation buffer Sambrook etal 1989 or the buffer provided with the commercial enzyme Notl Nru l Spe 1 Xba and other restriction enzymes e LB amp plates 50 mM NaCl autoclaved e Materials for purifying plasmid from E colitransformants Grow n Glow Yeast plasmid isolation kit can be used as well MoBiTec order 2069 1 pGNG2 12 2002 gt 15 MoBi Tec 16 7 1 3 2 Annealing of Sense Antisense Oligonucleotides Construction of the bait element for cloning into the GFP reporter vector 1 For each construct planned mix 0 1 pg of sense strand and 0 1 pg of antisense strand oligonucleotide of the bait element in 10 pl of 50 mM NaCl Anneal the bait element oligonucleotides by heating at 70 C for 5 minutes and then slowly cooling to room temperature 30 minutes Completely digest 0 1 pg of the GFP reporter plasmid in a 20 pl double digest using an appropriate pair of restrictio
21. ids fused to the B42 activation domain or no fusion peptide at all keep sequencing beyond the stop codon Another larger open reading frame ORF for a peptide that interacts with the target elements in your reporter strain may be found which functions as a transcriptional activator In yeast genomic libraries where intercistronic regions are very short nontranslated gaps upstream of ORF inserts are most commonly found Due to the cloning of a portion of the 5 untranslated region of the mRNA along with the coding region in the cDNA such gaps can also occur in cDNA libraries If the library was built in a high level expression vector such as the prey plasmid a western blot analysis will reveal the presence and size of a B42 activaton domain fusion protein b It has been observed that the positive library clone is transcribed in the reverse orientation from a cryptic promoter within the ADH1 terminator Chien etal 1991 Your sequencing results would then fail to reveal any ORF in frame with the B42 activator coding region The expressed proteins apparently function as transcriptional activators as well as interact with the target elements c In some cases two different ORFs may be expressed as a fusion with the B42 activation domain even though a nontranslated gap comes between them due for example to occasional translational read through 3 Ifyou havea library clone that you believe encodes a transcriptional activator check
22. ience 263 802 805 Chien C T Bartel P L Sternglanz R and Fields S 1991 The two hybrid system A method to identify and clone genes for proteins that interact with a protein of interest Proc Nat Acad Sci USA 88 9578 9582 Chiocchetti A Tolosano E Hirsch E Silengo L and Altruda F 1997 Green fluorescent protein as a reporter of gene expression in transgenic mice Biochim Biophys Acta 1352 193 202 Crameri A Whitehorn E A Tate E and Stemmer W P C 1996 Improved green fluorescent protein by molecular evolution using DNA shuffling Nature Biotechnol 14 315 319 Cubitt A B Heim R Adams S R Boyd A E Gross L A and Tsien R Y 1995 Understanding improving and using green fluorescent proteins Trends Biochem Sci 20 448 455 Dalton S and Treisman R 1992 Characterization of SAP 1 a protein recruited by serum response factor to the c fos serum response element Cell 68 597 612 Durfee T K Chen L Yeh S H Yang Y Kilburn A E Lee W H and Elledge S J 1993 The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit Genes Dev 7 555 569 Ghosh S Selby M J and Peterlin B M 1993 Synergism between Tat and VP16 in trans activation of HIV 1 LTR J Mol Biol 234 610 619 Holm C 1993 A functional approach to identifying yeast homologs of genes from oth
23. if the protein has a nuclear localization tag as many DNA transcrip pGNG2 12 2002 gt 21 MoBi Tec 22 tional activators have You may achieve a crude result by observing the pre dicted amino acid sequence and comparing it to known nuclear localiza tion sequences but of course the best evidence for a nuclear localization domain is functional evidence For example this could be analyzed by trans ferring the insert to an expression vector that will generate a fusion of the protein with a cellular localization tag such as the green fluorescent protein Gel shift DNA binding assay with protein extracts When preparing yeast protein extracts for use in an electrophoretic mobility shift assay EMSA use a procedure that will yield native proteins The following procedure is sum marized from Arndt ef al 1987 a Prepare an overnight culture of the yeast transformant in DOB TRP URA Glu medium to keep selection on the prey library plasmid The should be 1 0 b Centrifuge 100 ml of the culture Discard the supernatant and resuspend the pellet in 400 pl of extraction buffer 0 1 M Tris HCl pH 7 5 0 2 M NaCl 0 01 MB mercaptoethanol 20 glycerol 5 mM EDTA and 1 mM PMSF c Transfer cell suspension to a pre chilled glass tube and add an equal volume of glass beads Place sample on ice and vortex vigorously for 10 minutes not including pause times to allow for sample cooling d Allow glass beads to set
24. in diameter into 1 ml of YPD medium 2 Vortex vigorously for 2 minutes to disperse any clumps 13 14 15 16 Transfer enough overnight culture to produce an OD Incubate at 30 C for 3 hours with shaking at 230 rpm The OD Transfer 100 vl of this cell suspension into a flask containing 5 ml of YPD medium Incubate at 30 C for 16 18 hours with shaking at 250 rpm until stationary phase is reached gt 1 5 0 2 0 3 into 50 600 ml of YPD medium go Will be 0 5 0 1 Centrifuge the culture in 50 ml tubes at 2000x g for 5 minutes at room temperature Discard the supernatant and vortex to resuspend each cell pellet in 5 ml of TE buffer Centrifuge the cells again at 2000x g for 5 minutes at room temperature Discard the supernatant and resuspend the cell pellet in 300 pl of freshly prepared sterile 1x TE LiAc Mix well by vortexing n a sterile 50 ml tube add 0 2 1 pg of GFP reporter bait plasmid and 50 pg of carrier DNA and mix well Very important that the carrier DNA is denaturated several times by boiling in a water bath Add 250 pl of competent cells to the step 11 DNA mixture and mix well by vortexing Nas adada nda Add 1 2 ml of sterile PEG LiAc to the transformation mixture Vortex at high speed for 10 seconds to mix well Incubate at 30 C for 30 minutes with shaking at 200 rpm Add 100 pl of DMSO and mix well by gentle inversio
25. ized 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 Drop Out Base DOB medium or DOBA with Agar is a 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 Complete Supplement Mixture CSM 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 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 medium Just add water and autoclave ready For order informations see Appendix III pGNG2 12 2002 gt 11 MoBi Tec 5 1 1 Grow n Glow Yeast Growth Media Notes DOB Drop Out Base DOBA Drop Out Base with Agar TRP signifies medium lacks tryptophan URA signifies medium lacks uracil a YPD 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 the YPD broth bag MoBiTec order 4001 1 into a 0 51 flask add 500 ml H O Autoclave Ready for
26. ma e Sterile 1x PEG LiAc solution prepare immediately prior to use from 10x stocks 100 DMSO dimethylsulfoxide Sigma D 8779 1x TE buffer prepare from 10x TE buffer 100 mM Tris pH 7 5 10 mM EDTA e Sterile glass rod bent pasteur pipette or 5 mm glass beads for spreading transformation mixtures on plates Note The Yeast Transformation Systems MoBiTec order 2200 1 or 2100 1 contains all the solutions required for yeast transformation optimized for use in the Grow n Glow Two and One Hybrid Systems pGNG2 12 2002 gt 17 Mo Bi Tec 18 7 3 2 Large Scale Yeast Transformation This step is very critical The number of recombinants transformed with the prey clones should be as high as possible We therefore highly recommend the use of the Grow n Glow High Efficiency Yeast Transformation kit MoBiTec order 2200 1 for obtaining the best results contains an optimized library transfor mation procedure Otherwise you can try the up scaled procedure below This protocol is scaled for screening gt 1x 108 independent clones 1 Inoculate several colonies 4 5 of the transformed GFP reporter bait strain into 1 ml of DOB URA Glu medium 2 Vortex vigorously for 2 minutes to disperse any clumps 3 Transfer this cell suspension into a flask containing 50 ml of DOB URA Glu medium A Incubate at 30 C for 16 18 hours with shaking at 250 rpm to stationary phase gt 1 5 5 Transf
27. n Do not vortex pGNG2 12 2002 Mo Bi Tec 17 Heat shock for 15 minutes in a 42 C water bath Swirl occasionally to mix 18 Chill on ice for 2 minutes 19 Centrifuge at 2000x g for 5 minutes at room temperature and remove supernatant 20 Resuspend cells in 200 pl of TE buffer 21 Plate the transformation mixture onto an 85 mm DOB URA agar plate 22 Incubate at 30 C for 2 3 days Store the plate at 4 C when growth is achieved Appendix l Competent E coli Cells and Transformation Competent Cells 1 Inoculate one colony of your E coli strain in 5 ml SOB medium Note Use a freshly streaked plate with your E coli strain of choice 2 Incubate 250 ml SOB with 2 5 ml of the overnight culture Incubate at 37 C while shaking at 200 rpm Measure the OD after 2 2 5 hours Once a density of 0 4 0 5 OD is reached place cells on ice for 15 minutes It is very important to keep the temperature at 0 C unless other wise stated 3 Pellet cells by centrifugation Spin 10 minutes at 6000 rpm 3000 g at 4 C Discard the supernatant Resuspend in ice cold 80 ml RF buffer Place on ice for 20 minutes Spin 6000 rpm for 10 minutes at 4 C N QO UB Discard the supernatant Resuspend the pellet in ice cold 20 ml RF Il buffer 8 Aliquot the cells in 220 pl aliquots on ice Quick freeze immediately in liquid nitrogen or dry ice methanol Store at 80 C until use pGNG2 12 2002 gt 2
28. n between RAS and RAF and other protein kinases Proc Natl Acad Sci USA 90 6213 6217 Vojtek A Hollenberg S and Cooper J 1993 Mammalian Ras interacts directly with the serine threonine kinase Raf Cell 74 205 214 Wang M M and Reed R R 1993 Molecular cloning of the olfactory neuronal transcription factor Olf 1 by genetic selection in yeast Nature 364 121 126 Wilson T E Fahrner T J Johnston M and Milbrandt J 1991 Identification of the DNA binding site for NGFI B by genetic selection in yeast Science 252 1296 1300 Wu Lerchenko 1 and Filutowicz M 1994 Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein J Bacteriol 176 6795 6801 Yeh E Gustafson K and Boulianne G L 1995 Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila Proc Acad Sci USA 92 7036 7040 pGNG2 12 2002 Mo Bi Tec Appendix l Small Scale Yeast Transformation Reagents and Materials Required e YPD Medium chapter 5 1 1 a Appropriate sterile tubes and flasks Appropriate DOB URA agar plates chapter 5 1 1 b e Yeast strain EGY48 Herring testes carrier DNA Sigma e Sterile 1x PEG LiAc solution Prepare immediately prior to use from 10x stocks 100 DMSO Dimethylsulfoxide Sigma D 8779 1x TE buffer prep
29. n enzymes such as those recommended below Incubate at 37 C for 2 hours or as directed by the enzyme manu facturer GFP reporter NotI Nru and Spe Xba Electrophorese 2 pl of the digest on an 1 agarose gel to confirm that the plasmid has been efficiently linearized Mix 5 pl of the GFP reporter digested plasmid with 1 pl of annealed oligo Add 4 pl of H O Add 1 2 pl of T4 ligation buffer and 0 8 pl at least 0 8 units of T4 DNA ligase and incubate at room temperature for 4 hours Note Since the molar ratio of oligonucleotide to vector is 100 1 or greater no gel purification to remove the stuffer fragment is required Transform competent E coli cells with each construct using a standard method Sambrook et al 1989 Plate transformants on LB amp plates and incubate at 37 C overnight Prepare plasmid using any standard method that yields highly pure DNA Sambrook et al 1989 or use the MoBiTec plasmid isolation kit order 2069 1 for yeast which can also be used for plasmid DNA in E coli Check for inserts by sequencing across the junctions with the GFP reporter primer 7 2 Construction of the GFP Reporter Bait Strain We recommend a two step transformation procedure instead of co introducing the GFP reporter bait and prey plasmid library vectors A small scale transfor mation procedure is performed to transform the GFP reporter bait construct into the EGY 48 strain Use the
30. n of potential positives under UV light no time consuming B galactosidase lift assays as essential in most conventional assays antibodies to HA tag fused downstream of B42 permit simple immunoprecipitation of potential positives finds potentially toxic protein by screening with galactose inducible expression libraries pGNG2 12 2002 Mo Bi Tec auab 4 0 u ul l uonduosueu 4 5 Sulpulq YNA 4 u ul f 4918 uonduosueu ou Auojoo 1 09 1 99 you s Auojoo Kaad VNQ 01 pulg jou s op ulajoid K445 VNQ Aeud Z yg ul s4S pliqAH JOJOSA UUM POWJOJSULL Koid ules 1SE A ddo u ul l 496104 yoeoiddy piq H U0 yL UQ AAO O U AA046 BUY JO M AIAQ DYDWBYDS pGNG2 12 2002 MoBi Tec 4 Kit Components Vectors Primer Host Strain pGNG2 GNGprimer Yeast strain EGY48 pJG4 5 5 PREYprimer pGNG2 p53 3 PREYprimer pJG 4 5 p53 Re dissolve lyophilized vector DNA in TE buffer pH 8 0 Order information see Appendix III 4 1 DNA Vectors 4 1 1 GFP Reporter Vector Cloning site for One Hybrid Not I Nru VSpe GAL1 10 promoter The reporter plasmid pGNG2 is suited for one hybrid screens with the reporter gene GFP This GFPuv gene is driven by a GAL1 10 minimal prom
31. o amplify clone by PCR 4 2 3 3 PREYprimer 5 GCC GAC AAC CTT GAT TG 3 3 primer used to determine the identity of positive clones in pJG4 5 can also be used with 5 primer to amplify clone by PCR PCR is a process covered by patents owned by Hoffmann La Roche Use of this process requires a licence 4 3 Yeast Strain The provided yeast strain is a high sensitivity strain and has the following genotype S cerevisiae EGY48 MATa trp1 his3 ura3 leu2 6 LexAop LEU2 It is frozen in YPD medium containing 15 glycerol and can be stored indefinitely at 70 C pGNG2 12 2002 Mo Bi 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 Appendix Ill Similar items from other sources may be interchangeable 1 Grow n Glow Yeast Growth media see chapter 5 1 2 Grow n Glow Bacterial Growth media see chapter 5 1 3 Ampicillin Boehringer Mannheim order 835 269 4 Grow n Glow Yeast Transformation kits MoBiTec order 2100 1 and 2200 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 one hybrid systems Rich medium YPD and the standard Wickerham yeast nitrogen base with carbon source optim
32. oter The 6 51 kb plasmid contains a URA3 selectable marker 2 ym origin to allow propagation in yeast and the ampicillin resistance gene Amp and origin of replication pBR ori for propagation in Escherichia coli pGNG2 has unique restriction sites for NotI Nru I and Spe I suitable for cloning a DNA target element of interest for a one hybrid screen pBR ori GFPuv ADH1 terminator pGNG2 6541 bp 2um ori pGNG2 12 2002 Mo Bi Tec 4 1 2 Prey plasmid pJG4 5 The prey plasmid pJG4 5 Crameri etal 1996 is used to express cDNAs or other coding sequences inserted into the EcoR and Xho sites as translational fusions to a cassette consisting of the pUC ori SV20 nuclear localization sequence the Prey Plasmid 88 residue acidic activator B42 acid 6449 bp blob and the HA hemagglutinin epitope 2um ori tag Fusion protein expression is control m led by the GAL inducible promoter thus transcription levels are very low in the presence of glucose and high with galac tose For selection in yeast the vector cloning site for cDNA library contains the TRP1 tryptophan marker EcoR Xho and the 2 pm origin of replication for propagation in E coli an ampicillin resist ance Amp and pUC origin are present B42 cassette ADH terminator pJG4 5 polylinker 5 CCC GAA TIC GGC CGA CIC GAG AAG 3 Ecor Xho Polylinker of the vector indicating the open reading frame The shown restriction endon
33. oter region controlling expression of the GFPuv reporter in the GFP reporter vector A gene encoding a potential DNA binding protein is cloned in the prey plasmid so that it is expressed as a fusion to a B42 activator peptide Interaction of the DNA binding protein with the DNA element brings the B42 activator peptide into a position that initiates transcrip tion of the GFPuv reporter gene Thus colonies with DNA binding proteins that interact with the DNA element of interest are glowing brightly green which is easily seen by eye under UV illumination 300 nm 1 3 Green Fluorescent Protein GFP The GFP of the jellyfish A victoria is activated in vivo by an energy transfer via the Ca stimulation of the photoprotein aequorin Crameri ef al 1996 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 Chalfie etal 1994 and Cubitt et al 1995 It maximally absorbs light at 395 nm and has an emission peak at 509 nm The nonsubstrate requirement for GFP activity makes this protein an attractive reporter for gene expression studies and pGNG2 12 2002 gt 5 MoBi Tec this utility was initially demonstrated in both prokaryotes Escherichia coli and eukaryotes Caenorhabditis elegan
34. row n Glow library hybrid protein interacts with your bait element the transcription of the GFPuv gene is activated The interaction can be pGNG2 12 2002 Mo Bi Tec verified under UV light in a darkroom where colonies with DNA protein interactions will fluoresce green Our experience is that the Grow n Glow GFP One Hybrid System has a low incidence of false positives Next Grow n Glow library plasmids are isolated from the positive transformants and characterized by sequencing Finally DNA binding should be confirmed by independent methods The Grow n Glow GFP One Hybrid System can also be used to map the DNA binding domain of a known DNA binding protein The difference to the procedure described above is that instead of transforming the reporter bait strain with a library activation domain fusions are prepared with gene segments corresponding to specific domains of the known DNA binding protein These constructs are transformed into the reporter bait strain The GFPuv reporter is used and the GFPuv activity can be quantified using a liquid assay commercially available 1 2 Background Information The yeast one hybrid system Wang and Reed 1993 a modified version of the two hybrid system rapidly identifies DNA binding proteins from either cDNA libraries or known gene sequences The one hybrid system exploits the bi modular structure of eukaryotic transcriptional activators A DNA binding element of interest is cloned into the prom
35. s Chalfie et al 1994 It has subsequently been used to monitor gene expression in many organisms including mouse Chiocchetti et al 1997 Drosophila Yeh et al 1995 zebrafish embryos Meng ef al 1997 Arabidopsis Wilson ef al 1991 and yeast Niedenthal et al 1996 In addition to the non invasiveness of GFP detection long wave UV light the protein is very stable non toxic and resistant to photobleaching These properties make GFP a viable alternative to traditional reporter genes such as B galactosidase LacZ B glucuronidase GUS chloramphenicol acetyltransferase CAT or firefly luciferase which require substrate for their detection Several modifications of the wild type GFP cDNA have been engi neered with optimized codon usage improved fluorescence activity and red or blue shifted variants with altered excitation maxima intended for fluorescence microscopy Cubitt etal 1995 The A victoria GFP variant GFPuv used in this one hybrid system is optimized for maximal fluorescence by UV light excitation 360 400 nm making it fluoresce 18 times brighter than wild type GFP while retaining identical excitation and emission wavelength maxima 2 Advantages of the Grow n Glow One Hybrid System isolates genes for novel DNA binding proteins maps residues and regions responsible for DNA binding non invasive chemical free and cost free assay positive colonies fluoresce bright green therefore one step selectio
36. solated from yeast as described above 1 Isolate plasmids from four E coli colonies using any method that produces pGNG2 12 2002 gt 19 MoBi Tec 20 highly pure DNA Use the Grow n Glow Yeast Plasmid Isolation kit MoBiTec order 2069 1 for this purpose or consult Sambrook ef al 1989 Identify the E coli colonies containing the prey plasmid by restriction enzyme analysis by sequencing with the 5 PREYprimer or by colony PCR amplification using the 5 PREYprimer and 3 PREYprimer see next chapter Note PCR using the 5 PREYprimer and 3 PREYprimer with the prey vector carrying no insert as template DNA results in a 125 bp DNA fragment 7 3 5 Prey Plasmid Identification by PCR 1 Inoculate 4 colonies from each plate in 2 ml LB Amp medium Grow overnight at 37 C 5 pl culture are transferred to a PCR tube centrifuged 5 seconds at maximal speed Discard supernatant Add 30 pl PCR mix and mix PCR mix 20 pmole 5 PREYprimer 20 pmole 3 PREYprimer 3 pl 10x dNTP 2 5 mM dNTP 3 pl 10x Taq DNA polymerase buffer 1 pl Taq DNA polymerase 2 5 U pl H O up to 30 vl Run the reaction in a thermal cycler as follows 1 cycle 2 minutes 95 C 20 cycles 30 seconds 95 C 30 seconds 60 C 120 seconds 72 C 1 cycle 5 minutes 72 C Load the PCR reactions on an agarose gel Clones with an amplified insert contain a prey plasmid with an insert Go back to the cultures see step 1 Purify the t
37. tle then transfer all available liquid to another pre chilled glass tube e Add 200 pl of extraction buffer to the liquid add an equal volume of glass beads and vortex again as described above f Separate the liquid from the glass beads by centrifugation Note One way to do this is to punch a pinhole in the microcentrifuge tube and nest this tube inside another tube before adding the sample Upon centrifug ing the liquid will flow through to the collection tube leaving the beads behind g Freeze the liquid quickly in liquid nitrogen and store it at 70 C h The protein yield is typically 10 20 mg ml Use 2 5 pl in the EMSA Perform in vitro translation and a DNA binding assay Wu ef al 1994 A comparison of the gel shift results between the wild type bait DNA element and a mutant bait DNA element may prove excellently instructive pGNG2 12 2002 Mo Bi Tec 7 5 Troubleshooting Guide 7 5 1 Excessive Background Growth and Fluorescence on Library Screening Medium Solution 1 Check to make sure that you have prepared the selection medium correctly Solution 2 You may have used too high amounts of the Grow n Glow prey plasmid for transformation Perform a new transformation with lower amounts of the prey plasmid Solution 3 Your inserted bait element may be interacting with yeast endogenous transcrip tional activators lt may be necessary to redesign the target element and construct a new GFP reporter b
38. ucleases are only located in the polylinker 4 1 3 Control Plasmids GFP positive control vector pGNG2 p53 insert 3 x p53 consensus sequence Not I Spe GAL1 10 promoter The GFP positive control reporter vector pGNG2 p53 consists of pGNG2 and 3 tandem repeats of the mouse p53 consensus sequence cloned into the Not Spe sites of the pGNG2 MCS upstream of the Gall 10 promoter pBR ori GFPuv ADH1 terminator Amp p6NG2 p53 6608 bp pGNG2 p53 serves as positive control in combination with the prey control vector pJG4 5 p53 which carries the mouse p53 cDNA 2um ori URA3 pGNG2 12 2002 gt 9 Mo Bi Tec 10 Prey control plasmid pJG4 5 p53 The prey control plasmid pJG4 5 p53 consists of pJG4 5 and the sequence of the mouse p53 cDNA cloned into the EcoR 1 Xho sites of pJG4 5 which is fused to the B42 activation domain pUC ori pJG4 5 p53 7431 bp 2um ori GAL1 promoter gt ADH terminator B42 cassette insert p53 cDNA 4 2 Primer EcoR Xho 4 2 1 GNGprimer 5 GCC CAA TAC GCA AAC CGC CT 3 This sequence is situated 38 bp upstream of the unique restriction site Used to verify bait DNA introduced within the cloning sites of pGNG2 by sequencing the construct 4 2 2 5 PREYprimer 5 CTG AGT GGA GAT GCC TCC 3 5 forward primer used to determine the reading frame and identity of positive clones in pJG4 5 can be used with 3 primer t
39. y Synthesis 15 7 1 3 Tandem Copies of Bait Upstream of GFP Reporter Cen s l d 15 7 1 3 1 Reagents and Materials 15 7 1 3 2 Annealing of Sense Antisense Oligonucleotides 16 7 2 Construction of the Reporter Bait Strain 16 7 3 Screening a Grow n Glow cDNA Library for DNA Binding Protein Genes 17 7 3 1 Reagents and Materials Required 17 7 3 2 Large Scale Yeast Transformation 18 7 3 3 Plating the Transformations 19 7 3 4 lsolating the Prey Plasmid from Putative Positive Yeast Colonies jie poni 19 7 3 5 Prey Plasmid Identification by PCR 20 pGNG2 12 2002 7 4 Confirmation of DNA Binding Activity 21 7 5 Troubleshooting Gulide 23 7 5 1 Excessive Background Growth and Fluorescence on Library Screening Medium 23 7 5 2 Low Transformation Efficiency when Screening a Grow n Grow Library 23 7 5 3 False Negative Results 24 8 Literature v La 028 Appendix I Small Scale Yeast Transformation Procedure 27 Reagents and Materials Required
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