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Cloning into pPIC9K - Thermo Fisher Scientific

1. Overview Introduction Description Note Introduction Multiple copy integration of recombinant genes in Pichia has been demonstrated to increase expression of the desired gene in some cases Brierley et al 1994 Clare et al 1991a Cregg et al 1993 Romanos et al 1991 Scorer et al 1993 Scorer et al 1994 Thill et al 1990 Vedvick et al 1991 The pPIC9K vector included in this kit allows isolation of multicopy inserts by an in vivo method in order to test whether increasing the copy number of your recombinant gene will lead to a subsequent increase in secreted protein expression This in vivo method utilizes resistance to Geneticin G418 sulfate to screen for possible multicopy inserts The pPIC9K vector is identical to pPIC9 except for the presence of the kanamycin resistance gene for in vivo screening of multiple copy inserts pPIC9K is functional in Pichia strains GS115 and KM71 Additional features of the pPIC9K vector include e 9276 bp fusion vector e Four unique restriction sites for cloning in frame with the a factor secretion signal SnaB I EcoR I Avr IL Not I e Secreted expression of your gene using the o factor secretion signal e For expression your gene must be cloned in frame with the initiation codon of the signal sequence e HIS4 selection in Pichia e For gene replacement at AOX1 in GS115 linearize with Bg II generates Hist Mut e For insertion at AOX1 in G
2. Xcm I 699 2s 2s Aat II 9102 Tth III I 7034 Bgl ut 2 6875 Dra It 414 6713 6855 8046 8065 8757 Sal I 3178 BspE I B 3845 Restriction sites are outside the AOX1 sequences in the vector backbone but they are close enough for efficient recombination to occur tRestriction sites are used to generate gene replacements at AOX1 in GS115 only Continued on next page Transformation into Pichia Continued Controls Transformation into Pichia We recommend that you include the following controls when transforming Pichia The parent vector linearized in the same manner as your construct This will be used as a control to confirm integration via PCR see the Pichia Expression Manual for a protocol and as control for background in expression analysis and quantitative dot blots or Southern analysis Remember to also isolate a Hist transformant with just one copy of your gene inserted Most of the His transformants created by transforming with recombinant pPIC9K will only have one copy Make sure that the transformant you pick is only resistant to 0 25 mg ml Geneticin and that it has the same Mut phenotype as the putative multimeric recombinants you are testing This recombinant will be used as a control to compare expression levels with multiple copies of your expression cassette and as a control for single copy for quantitative dot blot or Southern analysis
3. Because of the genetic linkage between the kanamycin gene and the expression cassette PAOX1 and your gene of interest one can infer that Geneticin resistant clones contain multiple copies of your gene Secreted protein expression may increase because of a gene dosage effect Thus the presence of the kan gene on pPIC9K can be used as a tool to detect pPIC9K transformants that harbor multiple copies of your gene The graphic below shows multiple insertion of your expression cassette linked to the kan gene Expression Cassette 1 V 2nd Insertion Event ion 5 __AOXM or aox1 ARC4 TT 3 ES 5 Paoxyg eneofinterest TT HIS4 Kan jaa Cassette 1 Expression Cassette 2 J 3rd Insertion Event etc Continued on next page Overview Continued Screening on Geneticin Alternatives for Generating Multicopy Inserts Direct selection of Geneticin resistance in yeast does not work well because newly transformed cells need time to express sufficient amounts of the resistance factor Since yeast grow much more slowly than bacteria significant numbers of recombinant yeast are killed before they accumulate enough of the resistance factor to survive direct plating on antibiotic Do not use Geneticin resistance as a selectable marker The procedure to generate Geneticin resistant clones requires an initial selection of His transformants followed by a screen for varying levels of Geneticin resistance Resistance to Geneticin
4. C A Buckholz R G Clare J J and Romanos M A 1993 The Intracellular Production and Secretion of HIV 1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris Gene 136 111 119 Scorer C A Clare J J McCombie W R Romanos M A and Sreekrishna K 1994 Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High level Foreign Gene Expression Bio Technology 12 181 184 Strathern J N and Higgins D R 1991 Recovery of Plasmids from Yeast into Escherichia coli Shuttle Vectors In Guide to Yeast Genetics and Molecular Biology C Guthrie and G R Fink eds Methods in Enzymology J N Abelson and M I Simon eds Volume 194 Academic Press San Diego CA Thill G P Davis G R Stillman C Holtz G Brierley R Engel M Buckholz R Kinney J Provow S Vedvick T and Siegel R S 1990 Positive and Negative Effects of Multi Copy Integrated Expression in Pichia pastoris International Symposium on the Genetics of Microorganisms 2 477 490 Vedvick T Buckholz R G Engel M Urcan M Kinney J Provow S Siegel R S and Thill G P 1991 High level Secretion of Biologically Active Aprotonin from the Yeast Pichia pastoris J Ind Microbiol 7 197 201 Wach A Pick H and Phillippsen P 1994 Procedures for Isolating Yeast DNA for Different Purposes Protocol 3 In J R Johnston ed Molecular Genetics of Yeast A Practical Approach IRL Press
5. 10 days for authorization to return the unused Pichia expression products and to receive a full refund If you do agree to the terms of this license agreement please complete the User Registration Card and return it to Life Technologies before using the product Life Technologies Corporation Life Technologies grants you a non exclusive license to use the enclosed Pichia expression vectors Expression Vector for academic research or for evaluation purposes only The Expression Vectors are being transferred to you in furtherance of and reliance on such license You may not use the Expression Vectors for any commercial purpose without a license for such purpose from Research Corporation Technologies Inc Tucson Arizona Commercial purposes include any use of Expression Products or Expression Vectors in a Commercial Product any use of Expression Products or Expression Vectors in the manufacture of a Commercial Product any sale of Expression Products any use of Expression Products or the Expression Kit to facilitate or advance research or development directed to a Commercial Product and any use of Expression Products or the Expression Kit to facilitate or advance any research or development program the results of which will be directly applied to the development or manufacture of a Commercial Product Expression Products means products expressed with the Expression Kit or with the use of any Pichia expression vectors including the E
6. Feature Description Benefit 5 AOX1 An 1000 bp fragment containing Allows methanol inducible high level the AOX1 promoter expression in Pichia Targets plasmid integration to the AOX1 locus a factor Signal 269 bp fragment encoding the Allows secretion of desired protein into Tn903 which confers resistance to Geneticin in Pichia and kanamycin resistance in E coli Sequence a factor signal sequence for the medium secretion in Pichia MCS Multiple Cloning Site Allows insertion of your gene into the expression vector TT Native transcription termination Permits efficient transcription P P and polyadenylation signal from termination and polyadenylation of the AOX1 gene 260 bp mRNA HIS4 Pichia wild type gene coding for Provides a selectable marker to isolate histidinol dehydrogenase 2 4 kb Pichia recombinant strains and used to complement Pichia his4 strains 3 AOX1 Sequences from the AOX1 gene Targets plasmid integration at the AOX1 that are further 3 to the TT gene sequences 650 bp Amp Ampicillin resistance gene Allows selection replication and pBR322 origin E coli origin of replication maintenance in E coli Not I Unique restriction sites Permits linearization of vector for Bgl II efficient integration into the Pichia SacI genome and generation of either Mut or Sal I Mut recombinants kan Kanamycin resistance gene from Allows in vivo screening for multicopy inserts by incre
7. His transformants 1 Using a sterile spreader remove the top layer of soft agar containing the His transformants and place into a sterile 50 ml conical centrifuge tube 2 Add 10 to 20 ml of sterile water There should be a 2X volume of water above the settled agar Vortex vigorously for 1 2 minutes 3 Set centrifuge tube upright on bench and let agar pieces settle about 1 minute 4 Determine the cell density of the supernatant by using a hemacytometer You need at least 5 x 10 cells ml so you can plate 10 cells in 200 pl or less If the cells are too dilute transfer the liquid to a fresh tube and centrifuge the cells Resuspend the cell pellet in sterile water in a volume sufficient to give 5 x 10 cells ml 5 Plate 10 cells on YPD Geneticin plates containing Geneticin at a final concentration of 0 25 0 5 0 75 1 0 1 5 1 75 2 0 3 0 and 4 0 mg ml Use four plates for each concentration You may want to confirm the titer of the cells on YPD plates without Geneticin in order to calculate the percent of Geneticin resistant colonies you obtain for each Geneticin concentration and determine whether you are getting multimers at 1 10 of the transformants plated Prepare 10 10 and 10 dilutions of the pooled transformants using sterile water Plate 100 200 ul per plate 6 Incubate plates at 30 C and check daily Geneticin resistant colonies will take 2 5 days to appear while cells plated on YPD wi
8. This is a very important control as increasing the copy number of the desired gene does not always lead to increased expression of recombinant protein Refer to the Pichia Expression Manual for procedures to prepare Pichia for transformation transformation procedures selection of Hist recombinants and screening for Mut and Mut phenotypes if desired Once you have generated His transformants using recombinant pPIC9K proceed to In Vivo Screening of Multiple Inserts next page 11 In Vivo Screening of Multiple Inserts Introduction Methods to Screen for Geneticin Resistant Transformants Note 12 You will need as many His transformants as you can conveniently generate Recall that statistically 1 10 of the His transformants will have more than one insert This means that if the frequency of multicopy inserts is 1 you will have to screen 1000 His transformants to get 10 Geneticin resistant colonies to test This may require 1 5 plates containing His transformants It is not unusual to screen thousands of colonies Once you have Geneticin resistant colonies you can then characterize them for their Mut phenotype a cn P There are two methods used to screen His transformants for Geneticin resistance Method 1 is technically easier and screens a greater number of clones but is less reliable After initial selection of His transformants colonies are pooled and plated on YPD Geneticin plates conta
9. by streaking for single colonies Be sure to confirm the level of Geneticin resistance observed for each colony You may not find colonies resistant to 2 0 3 0 or 4 0 mg ml Geneticin Jackpot clones resistant to high levels of Geneticin are very rare You may have to screen thousands of His transformants in order to isolate colonies resistant to 2 4 mg ml Geneticin Analyze for the presence of your insert by PCR see the Pichia Expression System manual for a protocol PCR will only tell you if your gene is present It will not tell you how many copies of your gene are integrated or at which locus the integration occurred PCR can reasonably be done on 12 20 transformants Remember to include the vector only and original construct one copy controls in order to analyze your PCR experiment Since there is no guarantee that multiple copies will actually increase the amount of protein expressed most people elect to proceed directly to expression to see if any of these colonies over express their protein Be sure to include a single copy insert as a control Test all your Geneticin resistant colonies for their Mut phenotype so that you induce expression properly Refer to the Pichia Expression System manual for methods to express your protein Be sure to purify you clones by streaking for single colonies and making frozen glycerol stocks of all your Geneticin resistant colonies Always initiate expression studies from frozen s
10. please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Introduction Limited Use Label License No 74 Pichia Pastoris Expression System The Pichia Expression System is covered under the licenses detailed below The Pichia Expression System is based on the yeast Pichia pastoris Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology Industry Associates SIBIA and Phillips Petroleum for high level expression of recombinant proteins All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies RCT Inc Tucson Arizona Life Technologies has an exclusive license to sell Pichia expression kits and vectors to scientists for research purposes only under the terms described below Use of Pichia pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial license as detailed below Before using any Pichia expression product please read the following license agreement If you do not agree to be bound by its terms contact Life Technologies within
11. to estimate gene dosage It is very important to include DNA from the host strain alone GS115 or KM71 the host strain transformed with the parent vector pPIC9K and the host strain transformed with a vector containing one copy of your gene It is also a very good idea to make a probe to the HIS4 gene as an internal control for single copy in addition to a probe to your gene Note that if your gene inserts into his4 two copies of the HIS4 gene are created see Recombination and Integration in Pichia Pichia Expression Kit manual e Use standard procedures and solutions for Southern blotting as outlined in Molecular Cloning A Laboratory Manual Sambrook et al 1989 pages 9 31 9 58 e Isolate genomic DNA and quantify using fluorometry Be sure to eliminate RNA It is very important to load the same amount of DNA into each lane in order to accurately determine copy number e Probe your Southern blot with probes to both HIS4 and your gene Note that the point mutation in the his4 gene in the host strain will not interfere with hybridization if you make the probe complementary to the wild type gene e Use Bgl II to digest your DNA Clare et al 1991a Note that all multimers are NOT necessarily in a head to tail configuration Some multimers may be head to head and others tail to tail We recommend that you think about what products may be produced An expression cassette in the opposite orientation may produce a different band
12. 1994 You will need to prepare the following solutions e Minimal Medium MD MGY e Sterile water e SCED 1 M sorbitol 10 mM sodium citrate pH 7 5 10 mM EDTA 10 mM DTT e Zymolyase 3 mg ml stock solution in water Seikagaku America Inc e 1 SDS in water e 5M potassium acetate pH 8 9 e TE buffer pH 7 4 10 mM Tris HCl pH 7 4 1 mM EDTA pH 8 0 e 7 5 M ammonium acetate pH 7 5 e Phenol chloroform 1 1 v v 1 Grow the recombinant strain and the parent strain at 30 C to an ODsoo of 5 10 in 10 ml of minimal media such as MD or MGY recombinant or MDH or MGYH GS115 2 Collect the cells by centrifugation at 1500 x g for 5 10 minutes at room temperature 3 Wash the cells with 10 ml sterile water by centrifugation as in Step 2 1 Resuspend the cells in 2 ml of SCED buffer pH 7 5 Make this solution fresh 2 Add 0 1 0 3 mg of Zymolyase mix well before adding to the cells Incubate at 37 C for 50 minutes to achieve 80 spheroplasting 3 Add 2 ml of 1 SDS mix gently and set on ice for 5 minutes 4 Add 1 5 ml of 5 M potassium acetate pH 8 9 and mix gently 5 Centrifuge at 10 000 x g for 5 10 minutes at 4 C and save the supernatant Continued on next page 19 Pichia Genomic DNA Isolation Continued DNA Precipitation 20 1 Transfer the supernatant from Step 5 previous page and add 2 volumes of ethanol to the supernatant Incubate at room temperature for 15 minutes Ce
13. 200 ul per plate Incubate plates at 30 C and check daily Geneticin resistant colonies will take 25 days to appear while cells plated on YPD will take 2 3 days Proceed to Analysis of Results page 16 If you do not plate all of the cell suspension from either method above add sterile glycerol to 15 and freeze in convenient aliquots at 80 C You may thaw the aliquots and analyze for Geneticin resistant colonies at a later date In Vivo Screening of Multiple Inserts Continued Method 2 You will need three sets of two microtiter plates 6 total to screen 180 His recombinants Grow your clones to approximately the same cell density by successive inoculations to ensure that equivalent numbers of cells are spotted on Geneticin plates If you plated your transformants in top agar it may be necessary to extract them from the agarose and replate them on minus histidine plates see Pichia Expression System manual in order to pick colonies Remember to include controls for strain background and one copy of your gene For every 180 colonies you can expect to isolate 1 10 Geneticin resistant colonies 1 Using sterile technique add 200 ul YPD to each microtiter well 2 Inoculate each well of the first set of plates with a single His transformant using a sterile toothpick and stirring to resuspend cells 3 Cover the microtiter plate and incubate at 30 C for 2 days shaking not required 4 After 2 days take new mic
14. Invitrogen pPIC9K A Pichia Vector for Multicopy Integration and Secreted Expression Catalog no V175 20 Version G 03 June 2010 25 0106 Table of Contents Kit Contents anid Storage scene een RECTE RE Rn HI HERE Bauen tete ener euet v Acc ssory Products initi dee HU Saee GU ee ede Gl a ee HR m e Gere vi o 1 OVERVIEW cL Y 1 Materials ee deu oe adt shout doves o et SE 5 nn r 6 Cloning into pPIGOK aee eee E e en eee tede ese ese i E nr 6 Analysis of E coli Transformants zasi eeta esne pe ERa a eRe EEEa a EERTE na ne EOR EERE ARETES 9 Traristormation into Pichia manta eeu eed halen dete d tek mess 10 In Vivo Screening of Multiple Inserts nnnnnnnnsnenenensnseneerneneneeneneneneneneneneneeseneneneneneneenenenenenenenenenn 12 Analyzing Results vrees reeden deed dete eu 16 Troubleshooting uso einen tee iniit i ede db tru ti ee adit D st 17 Epid 18 Recipes oss eiea iaoee ote Sai oU Oh ERE REL BEER EOS RAI E es end 18 Pichia Genomic DNA Isolation sse tenete nennen a a tenent 19 Easy DNA Protocol for Isolation of DNA from Picltia i dud ists ctes ca tea iiri riesen sus 21 Determination of Copy Number of Multiple Integrants seen 22 Map and Features of pPICOK nueces een ede aire e ie i ann 24 Technical Support eee em e ned ts DER e ie iter tern 26 Purch ser Notification sess
15. Place the nitrocellulose filter face down on the paper and incubate for 5 minutes at room temperature 6 Remove the nitrocellulose filter and replace with two new 3MM sheets Soak with 10 15 ml of 2 x SSC Place the nitrocellulose filter face down on the 3MM paper and incubate for 5 minutes at room temperature Repeat 7 Bake nitrocellulose filters at 80 C or UV crosslink DNA to nylon The filters may be probed with a non radioactive labeled or random primed P labeled probe complementary to your gene Multi copy integrants can be identified by a strong hybridization signal relative to the single copy control Dot blots can then be quantified for copy number by densitometry of the film or blot or by using a scanner if radiolabeled Continued on next page Determination of Copy Number of Multiple Integrants Continued Southern Blot Analysis Controls General Guidelines For a detailed description of this technique as applied to Pichia pastoris see Clare et al 1991a It is very important to digest your DNA with the right restriction enzyme s to generate a blot of digested and gel separated genomic DNA Digestion of DNA from Pichia recombinants containing multiple copies will produce a band that will vary in intensity depending on the number of copies of your gene It is very important to include a control to show the intensity of a single copy gene The band intensities can be relatively quantified using densitometry
16. S115 or KM71 linearize with Sac I generates Hist Mutt in GS115 and Hist Mu in KM71 e For insertion at HIS4 linearize with Sal I generates Hist Mut in GS115 and Hist Mut in KM71 See page 10 for alternate restriction sites if your insert DNA has a Bg II Sac I or Sal I site There is no yeast origin of replication in any of the Pichia expression vectors included in this kit His transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome Continued on next page Overview Continued Frequency of Multicopy Inserts Generation of Multicopy Inserts in vivo 5 AOX1 or aox1 ARG4 TT 3 5 Paoxig Gene of Interest TT HIS4 Multiple plasmid integration events occur spontaneously in Pichia at a frequency between 1 and 10 of all His transformants The in vivo method allows you to screen for the His transformants that may have multiple inserts of your gene pPIC9K contains the bacterial kanamycin gene kan from Tn903 that confers resistance to Geneticin in Pichia Note that kan does not confer resistance to kanamycin in Pichia The level of Geneticin resistance roughly depends on the number of kanamycin genes integrated A single copy of pPIC9K integrated into the Pichia genome confers resistance to Geneticin to a level of 0 25 mg ml Multiple integrated copies of pPIC9K can increase the Geneticin resistance level from 0 5 mg ml 1 2 copies up to 4 mg ml 7 12 copies
17. Signal cleavage 1203 1204 Sna BI CTC GAG AAA AGA GAG GCT GAA GCT TAC Glu Lys Arg Glu Ala Glu Ala Tyr Leu Not I GCG GCC GCG AAT Ala Ala Asn Ala TCAGTTC AAGTTGGGCA CT a Factor primer site 1152 1172 TCC GCA TTA GCT GCT Ser Ala Leu Ala Ala ACG GCA CAA ATT CCG Thr Ala Gln Ile Pro TTA GAA GGG GAT TTC Leu Glu Gly Asp Phe AGC ACA AAT AAC GGG Ser Thr Asn Asn Gly AGC ATT GCT GCT AAA TAA TTCGCCTTAG KK pr TTACGAGAA GACCGGTCTT 3 AOX I primer site 1327 1347 CAAGAGG ATG GATACTT TTT 4 40X1 mRNA 3 end 1418 TCAGAAT GCCATTTGCC TGAGAGATGC TATTTGT AACCTATATA GTATAGGATT e The fragment containing the gene of interest must be cloned in frame with the secretion signal open reading frame e An initiating ATG is provided by the signal sequence Translation will initiate at the ATG closest to the 5 end of the mRNA e If your insert has a Bg II site see page 10 for alternate restriction sites to linearize your plasmid for Pichia transformation Analysis of E coli Transformants Introduction Analysis of Transformants Sequencing Recombinant Clones After Sequencing At this point you should have ligation reactions that you will transform by chemical means or electroporation into competent E coli cells TOP10 or equivalent using your method of choice 1 After transformation plate 10 ul and 100 ul of the transformation mix onto LB plates wit
18. The number of multiple copies will cause one or two bands depending on orientation in the Southern blot to increase in intensity once you are gt 2 copies e BglII digested DNA from GS115 and GS115 transformed with pPIC9K will produce bands of 2 8 kb from the genomic copy of HIS4 and 6 7 kb from the vector copy of HIS4 respectively when probed with a complementary fragment to HIS4 23 Map and Features of pPIC9K Map of pPIC9K The figure below shows the map of pPIC9K Details of the multiple cloning site and the a factor secretion signal are shown on page 8 The complete sequence of pPIC9K is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 26 ea G S o EcoR Avr Il Not 3 AOX1 TT Comments for pPIC9K 9276 nucleotides 5 AOX1 promoter fragment bases 1 948 5 AOX1 primer site bases 855 875 a Factor secretion signal s bases 949 1218 a Factor primer site bases 1152 1172 Multiple Cloning Site bases 1192 1241 3 AOX1 primer site bases 1327 1347 3 AOX1 transcription termination TT bases 1253 1586 HIS4 ORF bases 4514 1980 Kanamycin resistance gene bases 5743 4928 3 AOX1 fragment bases 6122 6879 Bgl II pBR322 origin bases 7961 7288 Ampicillin resistance gene bases 8966 8106 24 Map and Features of pPIC9K Continued Features of The table below describes the features of the pPIC9K expression vector pPIC9K
19. ased resistance to Geneticin Also allows selection for kanamycin resistance in E coli 25 Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our web site www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech support invitrogen com E mail E mail eurotechQinvitrogen com MSDS Certificate of Analysis Limited Warranty 26 jpinfo invitrogen com MSDSs Material Safety Data Sheets are available on our web site at www invitrogen com msds The Certificate of Analysis CofA provides detailed qual
20. at Oxford University Press New York NY pp 10 12 Zaret K S and Sherman F 1984 Mutationally Altered 3 Ends of Yeast CYC1 mRNA Affect Transcript Stability and Translational Efficiency J Mol Biol 177 107 136 2002 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 28 29 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
21. atible fragment into pPIC9K Refer to Ausubel et al 1990 pages 3 16 1 to 3 17 3 or Sambrook et al 1989 pages 5 10 to 5 13 for help with cloning The processing of the a factor mating signal sequence in pPIC9K occurs in two steps 1 The preliminary cleavage of the signal sequence by the KEX2 gene product with the final Kex2 cleavage occurring between arginine and glutamine in the sequence Glu Lys Arg Glu Ala Glu Ala where is the site of cleavage 2 The Glu Ala repeats are further cleaved by the STE13 gene product In Saccharomyces cerevisiae it has been noted that the Glu Ala repeats are not necessary for cleavage by Kex2 but cleavage after Glu Lys Arg may be more efficient when followed by Glu Ala repeats A number of amino acids are tolerated at site X instead of Glu in the sequence Glu Lys Arg X These amino acids include the aromatic amino acids small amino acids and histidine Proline however will inhibit Kex2 cleavage For more information on Kex2 cleavage see Brake et al 1984 There are some cases where Ste13 cleavage of Glu Ala repeats is not efficient and Glu Ala repeats are left on the N terminus of the expressed protein of interest This is generally dependent on the protein of interest Once you have decided on a cloning strategy you will need to prepare competent E coli cells for transformation before setting up your ligation reactions See Current Protocols in Molecular Biology Aus
22. conferred by the kanamycin gene present on pPIC9K is used as a screen not as a selection for multicopy integrants In addition to this vector for secreted expression Invitrogen has available two other vectors pPIC3 5K and pAO815 These vectors are designed for intracellular expression of recombinant proteins pPIC3 5K also uses Geneticin resistance to screen for multicopy inserts and uses the in vivo method pAO815 is used to construct multiple copies of your gene in vitro prior to transformation into Pichia Multiple copies are cloned in tandem into pAO815 and then are transformed into Pichia When His transformants are selected they will contain multiple copies of your gene A summary of each method is presented in the tables provided on the next page The best method is the one that works for your protein unfortunately there is no way to predict beforehand which method will work for you Continued on next page Overview Continued Methods for Generating Multicopy Inserts A summary of the advantages and disadvantages of the in vivo and in vitro method for generating multicopy inserts previous page is presented in the tables below The best method is the one that works for your protein unfortunately there is no way to predict beforehand which method will work for you In vivo Method pPIC9K and pPIC3 5K vectors Advantages Disadvantages Easier to initiate experiment because only one copy of you
23. eee pH eR e ede eere e etie lo deett 27 usi E ies 28 iii iv Kit Contents and Storage Shipping and pPIC9K is shipped on wet ice Upon receipt store pPIC9K at 20 C Storage Contents This kit contains 20 ug of pPIC9K vector supplied in suspension as 40 ul of 0 5 pg pl vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 Accessory Products Additional Products vi The products listed in this section are intended for use with the pPIC9K vectors For more information visit our web site at www invitrogen com or contact Technical Support page 26 Product Quantity Catalog no Original Pichia Expression Kit 1kit K1710 01 EasySelect Pichia Expression Kit 1 kit K1740 01 Easy DNA Kit 15 200 reactions K1800 01 TA Cloning Kit 20 reactions K2000 01 pPIC3 5K 20 ug each V173 20 pAO815 20 ug each V180 20 Pichia EasyComp Transformation Kit 1 kit K1730 01 Pichia Protocols 1 book G100 01 One Shot TOP10 chemically competent E coli 10 reactions C4040 10 20 reactions C4040 03 One Shot TOP10 Electrocompetent E Coli 10 reactions C4040 50 20 reactions C4040 52 TOP10 Electrocomp Kits 20 reactions C664 55 2x 20 reactions C664 11 Ampicillin Sodium Salt irradiated 200 mg 11596 027 Geneticin Selective Antibiotic powder 5g 11811 031 25g 11811 098 Geneticin Selective Antibiotic liquid 20 ml 10131 035 100 m1 10131 027
24. ell 4 Cool YPD to approximately 55 60 C and add appropriate volume of Geneticin stock see chart below Remember to also make several YPD plates without Geneticin O1 Mix well by swirling but be careful to minimize bubble formation 6 Pour agar solution into 10 cm petri plates Let plates harden invert and store bagged at 4 C Plates are stable for at least 6 months Final Geneticin mg ml ml Geneticin stock 250 ml YPD 0 25 0 625 0 50 1 25 0 75 1 875 1 00 2 5 1 50 3 75 1 75 4 375 2 00 5 0 3 00 7 5 4 00 10 0 Pichia Genomic DNA Isolation Introduction Solutions Needed Preparation Spheroplasting and Lysis The protocol below allows you to isolate DNA from the desired Hist recombinant and untransformed GS115 or KM71 The DNA isolated is suitable for Southern blot analysis dot slot blot analysis or genomic PCR See Current Protocols in Molecular Biology pages 13 11 1 to 13 11 4 Ausubel et al 1990 Guide to Yeast Genetics and Molecular Biology pages 322 323 Strathern and Higgins 1991 or Holm et al 1986 for other methods to isolate DNA from Pichia In addition to the protocol listed below we use our Easy DNA Kit page vi to isolate DNA from Pichia for PCR and quantitative dot slot blots See page 22 for this protocol Lastly there is a fast DNA isolation protocol for multiple samples 24 which has been reported Wach et al
25. erved in Pichia and other eukaryotic systems Henikoff and Cohen 1984 Irniger et al 1991 Scorer et al 1993 Zaret and Sherman 1984 If you have problems expressing your gene check for premature termination and AT rich regions It may be necessary to change the sequence in order to express your gene Scorer et al 1993 e The predicted protease cleavage sites for the a factor signal sequences are indicated in the multiple cloning site see page 8 e The open reading frame ORF of the mature gene of interest should be cloned in frame and downstream of the o factor signal sequence Continued on next page Cloning into pPIC9K Continued General Cloning Strategies Cloning Procedures Signal Sequence Processing Optimization of Signal Cleavage Bacterial Transformation Strategies generally fall into three different categories 1 Ligation of a compatible restriction fragment a Forced directional insertion involving the use of two different sites in the multiple cloning site b Ligation of the fragment with the same restriction end on both ends into a single compatible site 2 PCR amplification of the fragment containing the gene of interest in such a way that compatible restriction ends are generated for ligation into the appropriate vector 3 Direct cloning of an amplified fragment containing the gene of interest via the TA Cloning Kit Catalog no K2000 01 followed by subcloning of a comp
26. expression Multiple inserts are located at a single locus No need for a second drug resistance marker in the vector Materials Materials Needed Important For the procedures described in this manual you will need Manual from the Pichia Expression System Microbiological equipment Electrocompetent or chemically competent E coli must be recA endA for transformation You will need 3 4 tubes of competent cells per experiment For protocols to prepare competent E coli and transformation protocols see Current Protocols Ausubel et al 1990 or Molecular Biology A Laboratory Manual Sambrook et al 1989 Sterile water Phenol chloroform 3 M sodium acetate 100 ethanol 80 ethanol T4 Ligase 2 5 units ul 10X Ligation Buffer with ATP LB medium LB ampicillin plates 50 100 ug ml ampicillin 16 C 37 C and 65 C water baths or temperature blocks Geneticin YPD Geneticin plates see Recipes page 18 50 ml conical centrifuge tubes Hemacytometer 30 C and 37 C incubator Microtiter plates optional Procedures for transformation into E coli and Pichia analysis of recombinants and expression are described in the Pichia manual This manual is available for downloading from our web site at www invitrogen com or by contacting Technical Support page 26 Continued on next page Methods Cloning into pPIC9K Introduction General Considerations It is imp
27. h 50 100 pg ml ampicillin see Recipes page 18 and select ampicillin resistant colonies 2 Pick 10 ampicillin resistant transformants and inoculate into 2 ml LB medium with 50 100 pg ml ampicillin Grow overnight at 37 C with shaking 3 Isolate plasmid DNA by miniprep for restriction analysis and sequencing see below To sequence your construct in pPIC9K use the o factor and the 3 AOXI primer sequences see below 4 Make a glycerol stock of your desired clone for safekeeping by combining 0 85 ml of a overnight bacterial culture with 0 15 ml of sterile glycerol Mix by vortexing and transfer to a labeled storage tube Freeze the tube in liquid nitrogen or a dry ice ethanol bath and store at 70 C We strongly recommend that you sequence your construct before transforming into Pichia to confirm that your gene is in frame with the a factor secretion signal ATG We suggest using the a factor and 3 AOX1 primer sequences to sequence your construct Refer to the diagram on the previous page for the sequence and location of these primer binding sites For sequencing protocols refer to Unit 7 in Current Protocols in Molecular Biology Ausubel et al 1990 or Chapter 13 in Molecular Cloning A Laboratory Manual Sambrook et al 1989 Once you have cloned and sequenced your insert proceed to Transformation into Pichia page 10 You will need to generate enough plasmid DNA to transform Pichia 5 10 ug of each plasmid per each
28. h the most copies of your gene as successive multiple insertions are more rare Low isolation of His transformants Low transformation efficiency Using electroporation instead of spheroplasting may increase the transformation efficiency thereby allowing you to isolate more His transformants 17 Recipes Introduction YPD Geneticin Plates 18 Appendix See the current Pichia Expression manual for Pichia growth and selection media YPD Geneticin medium is described below Yeast Extract Peptone Dextrose Medium e 1 yeast extract e 2 peptone e 2 dextrose glucose e 1 5 agar e Variable amounts of Geneticin 10X D glucose 20 Dextrose Dissolve 200 g of D glucose in 1000 ml of water Autoclave for 15 minutes or filter sterilize The shelf life of this solution is approximately one year 100 mg ml Geneticin Geneticin is available separately from Invitrogen Catalog no 11811 031 Prepare 30 50 ml of 100 mg ml Geneticin stock solution in sterile water Filter sterilize and store frozen at 20 C You will use this solution to make YPD plates containing Geneticin at final concentrations of 0 25 0 5 0 75 1 0 1 5 1 75 2 0 3 0 and 4 0 mg ml For 250 ml 8 to 10 plates of a single Geneticin concentration 1 Combine 2 5 g yeast extract 5 g peptone and 5 g agar in 225 ml deionized water 2 Autoclave for 20 minutes on liquid cycle 3 Add 25 ml of 10X D and mix w
29. igh level Secretion Using Pichia pastoris Strains Containing Multiple Gene Copies Gene 105 205 212 Cregg J M Vedvick T S and Raschke W C 1993 Recent Advances in the Expression of Foreign Genes in Pichia pastoris Bio Technology 11 905 910 Henikoff S and Cohen E H 1984 Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces cerevisiae Mol Cell Biol 4 1515 1520 Holm C Meeks Wagner D W Fangman W L and Botstein D 1986 A Rapid Efficient Method for Isolating DNA from Yeast Gene 42 169 173 Irniger S Egli C M and Braus G H 1991 Different Classes of Polyadenylation Sites in the Yeast Saccharomyces cerevisiae Mol Cell Bio 11 3060 3069 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Romanos M A Clare J J Beesley K M Rayment F B Ballantine S P Makoff A J Dougan G Fairweather N F and Charles I G 1991 Recombinant Bordetella pertussis Pertactin p69 from the Yeast Pichia pastoris High Level Production and Immunological Properties Vaccine 9 901 906 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Plainview New York Scorer
30. ining increasing concentrations of Geneticin Method 1 is applicable to spheroplast or electroporation transformation methods Method 2 is technically more difficult and screens fewer numbers of clones but it is more reliable It involves growing clones in microtiter plates until all clones are at the same density The cultures are then spotted on the YPD Geneticin plates and scored for Geneticin resistance There is a tendency to isolate false positives when screening with Geneticin It is very important to purify your putative Geneticin resistant clones by streaking for single colonies on YPD and then confirming Geneticin resistance on YPD Geneticin plates For this reason we do not recommend replica plating as a method to screen for Geneticin resistance If you do elect to replica plate be sure to confirm Geneticin resistance The kanamycin resistance gene was cloned into pPIC9K with its native bacterial promoter The level of expression is very low You need to have multicopy integrants before you can begin to see resistance to Geneticin Continued on next page In Vivo Screening of Multiple Inserts Continued Before Starting Prepare 4 YPD plates containing each of the following concentrations of Geneticin 0 0 25 0 5 0 75 1 0 1 5 1 75 2 0 3 0 and 4 0 mg ml see Recipes page 18 Method 1 Use this procedure if you transformed using Pichia spheroplasts Start with Spheroplasts plates containing
31. inutes at 4 C remove ethanol and air dry 8 Resuspend the pellet in 50 ul TE containing 50 ug ml RNase A and incubate overnight at room temperature Quantify the amount of DNA We generally use 5 ul of this DNA solution in a 50 pl PCR reaction ae WN Bs 21 Determination of Copy Number of Multiple Integrants Introduction Quantitative Dot Blot Solutions Quantitative Dot Blot Procedure 22 You may wish to determine the actual number of gene copies in your Pichia recombinant You may either use quantitative dot blots or Southern hybridization to analyze gene copy number Brierley et al 1994 Clare et al 1991a Romanos et al 1991 Scorer et al 1993 Scorer et al 1994 This requires isolation of genomic DNA from Pichia recombinants transformed with the parent vector 0 copies of your gene pPIC9K containing 1 copy of your gene single copy control and the Pichia recombinants containing multiple copies of your gene Use the protocols detailed on the pages 19 and 21 to isolate genomic DNA You will need the following solutions 10 15 ml of each for each dot blot e 50 mM EDTA 2 5 mercaptoethanol pH 9 e 1mg ml Zymolyase 100T in water Seikagaku America Inc e 0 1 N NaOH 1 5 M NaCl 0 015 M sodium citrate pH 7x SSC 1X 0 15 M NaCl 0 015 M sodium citrate pH 7 e 3MM paper The following protocol is a summary of a rapid DNA dot blot technique to detect multiple integrants Romanos et a
32. ity control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications
33. l 1991 It is very important to spot equivalent numbers of cells onto filters in order to quantify copy number 1 Grow Mut or Mut transformants in individual wells of a 96 well microtiter plate in 200 pl of YPD broth at 30 C until all wells have approximately the same density This may necessitate several passages see page 16 for more details Alternatively individual transformants may be grown in culture tubes and the absorbance at 600 nm normalized with the addition of medium 2 Filter 50 ul of each sample onto a nitrocellulose or nylon filter placed into a dot slot blot apparatus using multi channel pipette Air dry filters 3 To lyse the cells on the filter you need to treat the filter with four solutions as follows place two sheets of 3MM paper in a tray and soak with 10 15 ml of 50 mM EDTA 2 5 mercaptoethanol pH 9 Make sure that the paper is uniformly soaked and that there are no puddles Place the nitrocellulose filter face down on the treated 3MM paper Incubate for 15 minutes at room temperature 4 Remove the nitrocellulose filter from the 3MM paper and replace the 3MM paper with two new sheets Soak with 10 15 ml of 1 mg ml Zymolyase 100T as described in Step 3 Place the nitrocellulose filter face down on the 3MM paper and incubate for 4 hours at 37 C 5 Remove the nitrocellulose filter from the paper and replace the paper with two new sheets Soak with 10 15 ml of 0 1 N NaOH 1 5 M NaCl 0 015 M sodium citrate pH 7
34. log no K1800 01 e Chloroform e Isopropanol e 70 or 80 ethanol e RNase A 1 Grow the recombinant strain and the parent strain at 30 C to an OD600 of 5 10 in 2 5 ml of minimal media such as MD or MGY recombinant or MDH or MGYH GS115 or KM71 2 Harvest 1 5 ml of the culture by centrifuging at maximum speed in a microcentrifuge for 1 2 minutes at room temperature 3 Resuspend cells in 1 5 ml TE and centrifuge as in Step 2 4 Resuspend cells in 1 ml fresh 1 M Sorbitol 100 mM EDTA 14 mM mercaptoethanol Vortex to resuspend 5 Add 1 5 ul of 3 mg ml Zymolyase to each tube of cells and incubate at 30 C for 1 hour 6 Centrifuge at 2 600 x g ina microcentrifuge for 8 minutes at room temperature It is important to centrifuge with less force as the cells are fragile because of digestion with Zymolyase 7 Gently resuspend cells in 200 ul fresh SCED and incubate at 37 C for 1 hour Add 350 ul Easy DNA Solution A to the cell suspension from Step 7 above vortex and incubate at 65 C for 10 minutes Add 150 ul of Easy DNA Solution B and vortex Add 600 ul chloroform and vortex Centrifuge at maximum speed for 20 minutes at room temperature Transfer the aqueous layer to a fresh tube add 600 ul isopropanol and mix by inversion Incubate at room temperature for 10 minutes Centrifuge sample at maximum speed for 20 minutes at 4 C Wash pellet with cold 70 or 80 ethanol centrifuge at maximum speed for 2 m
35. ntrifuge at 10 000 x g for 20 minutes at 4 C 3 Resuspend the pellet gently in 0 7 ml of TE buffer pH 7 4 and transfer to a microcentrifuge tube Gently extract with an equal volume of phenol chloroform 1 1 v v followed by an equal volume of chloroform isoamyl alcohol 24 1 Split the aqueous layer into two microcentrifuge tubes Add 1 2 volume of 7 5 M ammonium acetate pH 7 5 and 2 volumes of ethanol to each tube Place on dry ice for 10 minutes or at 20 C for 60 minutes Centrifuge at 10 000 x g for 20 minutes at 4 C and wash the pellets once with 1 ml of 70 ethanol Briefly air dry the pellets and resuspend each one in 50 pl of TE buffer pH 7 5 Determine the concentration of the DNA sample The two samples can be stored separately or combined and stored at 20 C until ready for use Easy DNA Protocol for Isolation of DNA from Pichia Introduction Solutions Needed Preparation of Cells DNA Isolation The method below was developed at Invitrogen to conveniently isolate DNA from Pichia pastoris You will need to prepare the following solutions e Minimal Medium MD MGY e TE buffer pH 7 4 10 mM Tris HCl pH 7 4 1 mM EDTA pH 8 0 e 1 M Sorbitol 100 mM EDTA 14 mM mercaptoethanol make fresh e Zymolyase 3 mg ml stock solution in water Seikagaku America Inc e SCED 1 M sorbitol 10 mM sodium citrate pH 7 5 10 mM EDTA 10 mM DTT make fresh e Easy DNA Kit Invitrogen Cata
36. ortant to clone your gene in frame with the a factor signal sequence Below are some guidelines to consider when developing a cloning strategy for this vector Refer to page 8 for the multiple cloning site of pPIC9K We recommend that you transform pPIC9K into E coli so that you have a permanent stock and a way to make more plasmid e Dilute 1 ul of the plasmid 1 ug ul to 10 100 pg ul using sterile water or TE buffer e Transform competent E coli with 1 2 ul of the diluted plasmid and select on LB with 50 100 ug ml ampicillin LB Amp The following are some general considerations applicable to pPIC9K e The codon usage in Pichia is believed to be the same as Saccharomyces cerevisiae e Many Saccharomyces genes have proven to be cross functional in Pichia e Plasmid constructions should be maintained in a recA endA mutant E coli strain such as TOP10 Electrocompetent TOP10 cells are available from Invitrogen page vi e The native 5 end of the AOX1 mRNA is noted in the multiple cloning site see page 8 This is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason e Translation termination is determined by either stop codons in the gene of interest or in the 3 AOX1 sequence The stop codons in the 3 AOX1 sequence are noted in the multiple cloning site see page 8 e The premature termination of transcripts because of AT rich regions has been obs
37. products in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Pichia Expression products in your control and so notify Life Technologies in writing You may contact Research Corporation Technologies at the following address Bennett Cohen Ph D Research Corporation Technologies 101 North Wilmot Road Suite 600 Tucson Arizona 85711 3335 Tel 520 748 4443 Fax 520 748 0025 27 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1990 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Brierley R A Davis G R and Holtz G C 1994 Production of Insulin Like Growth Factor 1 in Methylotrophic Yeast Cells United States Patent 5 324 639 Cavener D R and Stuart C R 1991 Eukaryotic Start and Stop Translation Sites Nucleic Acids Res 19 3185 3192 Clare J J Rayment F B Ballantine S P Sreekrishna K and Romanos M A 1991a High level Expression of Tetanus Toxin Fragment c in Pichia pastoris Strains Containing Multiple Tandem Integrations of the Gene Bio Technology 9 455 460 Clare J J Romanos M A Rayment F B Rowedder J E Smith M A Payne M M Sreekrishna K and Henwood C A 1991b Production of Epidermal Growth Factor in Yeast H
38. r gene is cloned into pPIC3 5K or pPIC9K before transforming into Pichia Qualitative screen Geneticin resistance roughly correlates with the number of copies of your gene Identifies the 1 10 of spontaneous His transformants that have multiple inserts Screening His transformants may involve more work because you will need thousands of His transformants to generate enough Geneticin resistant colonies to test Average size of vector is similar to other Pichia expression vectors The number of multiple inserts is unknown although this can be determined through Southern or dot blot analysis Multiple inserts are located at a single locus Screening on Geneticin is sensitive to the density of the cells and may result in the isolation of false positives In vitro Method pAO815 vector Advantages Disadvantages Quantitative construction of a defined number of multimers More work up front to clone defined number of multimers Most of the His transformants will contain the proper defined number of inserts Size of the vector may become quite large depending on the size of your gene and the number of copies you create Isolation of recombinants with multiple inserts is easier because most of the His transformants will contain multiple copies of your gene Rearrangements in E coli may occur In vitro construction allows step wise analysis of copy number effects on protein
39. rotiter plates and add 190 ul of YPD to each well Inoculate the second set of microtiter plates with 10 ul from the first set of microtiter plates by using a multi channel pipette Make sure the second set of plates is marked and oriented in such a way that you can keep track of wells 6 Cover and incubate the second set of plates overnight at 30 C 7 The next day repeat Steps 5 6 creating a third set of microtiter plates Note Successive growth and passage of the clones will bring them all to the same cell density 8 After incubation take the third set of plates and resuspend the cells in each well by pipetting up and down with a multi channel pipette set on 100 ul volume 9 Spot 10 pl from each well on YPD plates containing Geneticin at a final concentration of 0 0 25 0 5 0 75 1 0 1 5 1 75 2 0 3 0 and 4 0 mg ml Spot in a regular pattern using the multi channel pipette or a grid underneath the plate 10 Let the liquid soak in then incubate plates at 30 C and check after 2 3 4 or 5 days for Geneticin resistant clones Proceed to Analysis of Results next page Continued on next page 15 Analyzing Results Analysis of Results Vly N MENO Oo O RE NOTES Determination of Copy Number 16 There may be only a few Geneticin resistant colonies and they may be of different sizes but the colony morphology should be the same Pick all Geneticin resistant colonies and purify
40. thout Geneticin will take 2 3 days Proceed to Analysis of Results page 16 Continued on next page 13 In Vivo Screening of Multiple Inserts Continued Method 1 Electroporation Note 14 Use this procedure when electroporation was used to transform Pichia Transformants will not be plated in top agar Start with plates containing His transformants 1 Pipette 1 2 ml sterile water over the His transformants on each plate Use all the plates that have His transformants Resuspend the His transformants into the water by using a sterile spreader and running it across the top of the agar Be careful not to tear the agar Transfer and pool the cell suspension into a sterile 50 ml conical centrifuge tube and vortex briefly 5 10 seconds Determine cell density using a hemacytometer or spectrophotometer 1 ODsoo 5 x 107 cells ml Note any agar present will interfere with a spectrophotometer reading Plate 10 cells on YPD plates containing Geneticin at a final concentration of 0 25 0 5 0 75 1 0 1 5 1 75 2 0 3 0 and 4 0 mg ml You may want to confirm the titer of the cells on the YPD plates without Geneticin in order to calculate the percent of Geneticin resistant colonies you obtain for each Geneticin concentration and determine whether you are getting multimers at 1 10 of the transformants plated Prepare 10 10 and 107 dilutions of the pooled transformants using sterile water Plate 100
41. tocks not old plates If you find that your Geneticin resistant His recombinants significantly over express your protein you may wish to quantify the copy number of your gene Copy number may be analyzed by Southern or quantitative dot slot blots See pages 22 23 for information on these techniques It is very important to include genomic DNA isolated from Pichia recombinants transformed with pPIC9K alone and pPIC9K with a single copy of your gene as controls in order to evaluate your experiment Troubleshooting Introduction Review the information below to troubleshoot your experiments using pPIC9K Problem Cause Solution False positives Not enough levels of Geneticin resistance Colonies can appear to be Geneticin resistant but are actually not To prevent this purify your putative Geneticin resistant colonies and confirm the level of Geneticin resistance observed for each colony before proceeding further Low number of Geneticin resistant colonies isolated Not enough His transformants were screened The frequency of spontaneous multiple integration events occurs only at a rate of 1 10 You will need to screen thousands of His transformants to isolate an optimal amount of colonies Few recombinants with gene of interest Not enough His transformants were screened You will need to screen thousands of His transformants to isolate an optimal amount of recombinants wit
42. transformation Transformation into Pichia Introduction Linearization of Plasmid DNA Alternate Restriction Sites 10 At this point you will have your gene cloned in pPIC9K You should also have about 5 10 ug of each construct for each transformation into Pichia For methods to transform Pichia and select His transformants refer to the Pichia Expression System manual To linearize your construct prior to transformation into Pichia see below It is recommended that you linearize your vector in such a manner to generate both Mut and Muf recombinants It is possible that one phenotype will express your multicopy integrant better than the other To linearize pPIC9K containing one copy of your gene e BglII for replacement at AOX1 GS115 Mut e SacI for insertion at AOX1 GS115 Mut or KM71 Mut e SalI for insertion at HIS4 GS115 Mutt or KM71 Mut Use strain KM71 if you only want Muf recombinants If your insert contains any of the above restriction sites see the table below for alternate sites The table below describes alternate restriction sites for linearizing your construct before transformation into Pichia pPIC9K Note that an additional Stu I site was added with the inclusion of the kan gene eliminating the unique Stu I site in HIS4 Restriction 5 AOX1 3 AOX1 Vector backbone HIS4 gene Enzyme Sac I 209 Pme I 414 Bpu 1102 I 589
43. ubel et al 1990 or Molecular Biology A Laboratory Manual Sambrook et al 1989 for preparation of electrocompetent or chemically competent E coli or use your laboratory s procedure Continued on next page Cloning into pPIC9K Continued PAOX1 and Multiple Cloning Site of pPIC9K Special Considerations The sequence below shows the detail of the multiple cloning site and surrounding sequences AOXI mRNA S end 824 TTATCATCAT TATTAGCTTA C1 AAGCTTTTGA TTTTAACGAC TI CAACTAATTA TI TTT ACT GCA Phe Thr Ala CCA GTC AAC Pro Val Asn GCT GAA GCT Ala Glu Ala GAT GTT GCT Asp Val Ala TTA TTG TTT Leu Leu Ph GTT Val ACT Thr GIG Val GTT Val ATA It GAA GAA GGG Glu Glu Gly Eco RI l GTA GAA TTC Val Glu Phe ACATGACTGT TCC1 GCTAGATTCT AAT AGGCTTCATT TT TTTTTTGTCA GTA Val Te CCT Pro TTA Leu ACA ThE ATC Ile TTG Leu AAT Asn TCT Ser AGG Arg Start 949 5 AOXI primer site 855 875 rd ist a Bu TTCATAAT TGCGACTGGT TCCAATTGAC TTAACGAC AACTTGAGAA GATCAAAAAA a Factor Signal Sequence TCGAAGGAT CCAAACG ATG AGA TTT CCT TCA ATT Met Arg Phe Pro Ser Ile TTC GCA GCA TCC Phe Ala Ala Ser ACA GAA GAT GAA Thr Glu Asp Glu GGT Gly CCA u TAC TCA GAT yr Ser Asp TTT TCC AAC Pro Phe Ser Asn ACT ACT ATT GCC Thr Thr Ile Ala Ser Ile Ala Ala Lys W
44. xpression Vector or host strains Commercial Product means any product intended for sale or commercial use Commercial entities may conduct their evaluation for one year at which time this license automatically terminates Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding their desire for a commercial license Access to the Expression Kit and Vector must be limited solely to those officers employees and students of your institution who need access to perform the above described research or evaluation You must inform each such officer employee and student of the provisions of this license agreement and require them to agree in writing to be bound by the provisions of this license agreement You may not distribute any Expression Vector or host strain contained herein or in the Expression Kit to others even those within your own institution You may only transfer modified altered or original material from the Expression Kit or Vector to a third party following written notification of and written approval from Life Technologies so that the recipient can be licensed You may not assign sub license rent lease or otherwise transfer this license agreement or any of the rights or obligation there under except as expressly permitted by Life Technologies and RCT This license agreement is effective until terminated You may terminate it at any time by destroying all Pichia Expression

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