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pPIC3.5K - Thermo Fisher Scientific

1. Restriction 5 AOX1 3 AOX1 Vector backbone HIS4 gene Enzyme Sac I 209 B Pme lI 414 Bpu 11021 589 Xcm I 699 Aat II 8843 Tth I I 6775 zz Bgl II 2 6616 Dra It 414 6454 6596 7787 7806 Sal I 2919 BspEI zm 3580 pAOS15 Note that if more than one expression cassette is created in pAO815 most of the unique sites in the 5 AOXI region are now duplicated and no longer unique Restriction 5 AOX1 3 AOX1 Vector backbone HIS4 gene Enzyme Aat II 7535 Ee xc Tth I I 5467 Bgl II 2 5307 Sal I x 2863 Stu I 2948 BspEI 3580 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 Transforming Pichia Analysis of His Transformants Important We recommend that you include the following controls when transforming Pichia e The parent vector linearized in the same manner as your construct This is used as a control to confirm integration via PCR see the Pichia Expression Manual for a protocol and as control for background for the expression analysis and the quantitative dot blot
2. 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 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 to accurately determine copy number 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 If you used pPIC3 5K to generate multimers use Bgl II to digest your DNA Clare et al 1991a Note that if you used pPIC3 5K 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 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 2 copies If you used pAO815 to generate multimers use Bgl II and BamH I to digest the genomic DNA and release the multimer The molecular weight of the band should allow you to determine the number of multimers If this multimer is too large you may wish to digest with an enzyme like Sac I This will collapse the multimer into single fragment
3. 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 High 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
4. 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 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 withou
5. and air dry the pellet Resuspend pellet in 4 uL sterile water Save on ice if you plan to ligate your insert immediately or you can store at 20 C Proceed to Ligating Multimers into Linearized Vector next page Continued on next page pAO815 n Vitro Multimerization Protocol Continued Note Ligating Multimers into Linearized Vector Transforming E coli You may wish to combine the ligation reaction with the restriction enzyme digestion T4 ligase will retain most of its activity in all of the four New England BioLabs buffers Remember to add 1 mM ATP to the reaction to ensure ligase activity You are now ready to ligate the mixture of multimers generated in Step 10 above into dephosphorylated linearized vector 1 Setup the following ligation reactions Dephosphorylated vector page 17 Step 10 4 uL Expression cassette multimers Step 10 above 4uL 10X Ligation Buffer 1 pL T4 DNA Ligase 2 5 units WL 1 pL Total volume 10 uL For the vector only control Dephosphorylated vector 4uL Sterile water 4 uL 10X Ligation Buffer 1uL T4 DNA Ligase 2 5 units UL 1uL Total volume 10 uL 2 Incubate overnight at 16 C 3 You may store the ligation reactions at 20 C until ready to use or transform 1 10 uL of each ligation mix into competent E coli Note that the amount of the ligation mixture you transform depends on whether you use electrocompetent or chemically competent cells You may have to decrease t
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7. AOX 1 primer site 855 875 MM TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC pr AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA EROR I CAACTAATTA TTCGAAACGA GGAATTCGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA 3 AOX 1 primer site 1024 1044 GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT AOX1 mRNA 3 end 1115 ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT Special Considerations Transformation 10 For in vitro multimerization you need to analyze your insert for BamH I and Bgl II restriction sites If your insert has a BamHI or Bgl II site we recommend that you use the in vivo method pPIC3 5K to isolate multiple inserts of your gene For pAO815 the fragment containing the gene of interest should have a Kozak consensus sequence for proper translation initiation although this requirement is not as stringent in yeast For example ACC ATG G isa Kozak consensus sequence where the ATG corresponds to the initiating ATG for your gene of interest Cavener and Stuart 1991 Kozak 1987 and Kozak 1990 Be sure to analyze the 5 untranslated region of the mRNA for secondary structure formation Secondary structure in the mRNA has a negative effect on expression of the recombinant protein At this point you should have ligation reactions that you will transform by chemical means or electroporation into competent E coli cel
8. Buffer with ATP LB Amp plates 50 100 ng mL ampicillin 16 C 37 C and 65 C water baths or temperature blocks Continued on next page 15 pAO815 n Vitro Multimerization Protocol Continued Controls Digesting Recombinant pAO815 Producing Expression Cassettes for Multimerization 16 To evaluate your transformants and expression data later on we recommend transforming Pichia with pAO815 the parent vector and pAO815 containing one copy of your expression gene This will allow you to compare expression levels to see if multiple copies significantly increase the amount of protein produced Also if you elect to determine how many copies of your gene are in a recombinant by dot or Southern blot the strain with the parent vector will control for background hybridization and the strain with the single copy gene will provide a signal to normalize your data Set up two separate digestions of recombinant pAOS815 containing one copy of your gene 1 Double digest 1 2 ug recombinant pAO815 with 10 units each of Bgl II and BamHI Use a 20 uL reaction volume and digest for 1 2 hours at 37 C to release your expression cassette Proceed to Producing Expression Cassettes for Multimerization Step 1 2 Digest 2 ug recombinant pAO815 with 10 units of BamH I only Use a 20 uL reaction volume and digest for 1 2 hours at 37 C to linearize recombinant pAO815 Proceed to Dephosphorylating the Vector Step 1 The S N A
9. His transformants on each plate Use all the plates that have Hist 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 to 10 seconds Determine cell density using a spectrophotometer 1 OD 5 x 107 cells mL Note Any agar present will interfere with a spectrophotometer reading Plate 105 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 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 1075 10 6 and 107 dilutions of the pooled transformants using sterile water Plate 100 to 200 uL per plate Incubate plates at 30 C and check daily Geneticin resistant colonies will take 2 to 5 days to appear while cells plated on YPD will take 2 3 days Proceed to Analyzing the Results page 31 If you do not plate all of the cell suspension from either of the methods above Note add sterile glycerol to 15 and freeze in convenient aliquots at 80 C You may thaw the aliquots and analyze for Geneticin resistant co
10. P Gel Purification Kit available from Invitrogen see page 44 for ordering allows you to rapidly purify DNA fragments from regular agarose gels Alternatively you may use glass milk To use the S N A P Gel Purification Kit follow the steps below 1 Electrophorese your digest from Step 1 above on a 1 to 5 regular TAE agarose gel Note Do not use TBE to prepare agarose gels Borate interferes with the sodium iodide step below 2 Cutout the gel slice containing the PCR product and melt it at 65 C in 2 volumes of the 6 M sodium iodide solution 3 Add 1 5 volumes Binding Buffer Load solution no more than 1 mL at a time from Step 3 onto a S N A P column Centrifuge 1 minute at 3 000 x g in a microcentrifuge and discard the supernatant 5 If you have solution remaining from Step 3 repeat Step 4 Add 900 uL of the Final Wash Buffer Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow through 8 Repeat Step 7 9 Elute the purified DNA in 15 uL of sterile water Store on ice if proceeding immediately to Ligating the Expression Cassette page 18 Store at 20 C for long term storage Continued on next page pAO815 n Vitro Multimerization Protocol Continued Dephosphorylating Dephosphorylation is necessary to prevent self ligation of the vector the Vector 1 2 10 Take your digest from Digesting Recombinant pAOS15 Step 2 and phenol extract then ethanol precipitate
11. P4ox and your gene of interest one can infer that Geneticin resistant clones contain multiple copies of your gene Protein expression may increase because of a gene dosage effect Thus the presence of the kan gene on pPIC3 5K can be used as a tool to detect pPIC3 5K transformants that harbor multiple copies of your gene The graphic on the following page shows multiple insertion and linkage of the kan gene to your expression cassette Continued on next page Product Overview Continued 5 AOX1loraoxi ARG4 TT 3 5 Paoxig ene of Interest TT Kan HIS4 Expression Cassette 1 J 2nd Insertion Event 5 AOX1oraox1 ARG4 TT 3 expression arz n Paoxg Gene of interest TT j Kan HIS4 JAS aox Cassette 1 Expression Cassette 2 J 3rd Insertion Event etc Screening on Direct selection of Geneticin resistance in yeast does not work well because Geneticin 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 conferred by the kanamycin gene present on pPIC
12. 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 uL 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 Solutions Preparing Cells You will need to prepare the following solutions Minimal Medium MD MGY TE buffer pH 7 4 10 mM Tris HCl pH 7 4 1 mM EDTA pH 8 0 1M Sorbitol 100 mM EDTA 14 mM f mercaptoethanol make fresh Zymolyase 3 mg mL stock solution in water Seikagaku America Inc SCED 1 M sorbitol 10 mM sodium citrate pH 7 5 10 mM EDTA 10 mM DTT make fresh Easy DNA Kit to purchase see page 44 Chloroform Isopropanol 70 or 80 ethanol RNase A Grow the recombinant strain and the parent strain at 30 C to an OD of 5 10 in 2 5 mL of minimal media such as MD or MGY recombinant or MDH or MGYH GS115 or KM71 Harvest 1 5 mL of the culture by centrifuging at maximum speed in a microcentrifuge for 1 2 minutes at room temperature Resuspend cells in 1 5 mL TE and centrifuge as in Step 2 Resuspen
13. 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 Important 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 Before Starting Prepare 4 YPD plates containing 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 36 Continued on next page 27 pPIC3 5K n Vivo Screening of Multiple Inserts Continued Method 1 Spheroplasts 28 Use this procedure if you transformed Pichia spheroplasts Start with plates containing His transformants 1 Using a sterile spreader remove the top layer of the soft agar containing the His transformants and place into a sterile 50 mL conical centrifuge tube Add 10 to 20 mL of sterile water There should be a 2X volume of water above the settled agar Vortex vigorously for 1 to 2 minutes Set centrifuge tube upright on bench and let agar pieces settle about 1 minute Dete
14. day repeat Steps 5 and 6 creating a third set of microtiter plates Note Successive growth and passage of 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 multichannel pipette set on 100 uL volume 9 Spot 10 uL 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 Analyzing the Results next page Continued on next page pPIC3 5K n Vivo Screening of Multiple Inserts Continued Analyzing the Results EN BECO z Nous l Determining Copy Number 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 by streaking for single colonies Be sure to confirm the observed level of Geneticin resistance You may not find colonies resistant to 2 0 3 0 or 4 0 mg mL Geneticin Jackpot clones resistant to these high levels of Geneticin are very rare You may have to screen thousands of His transformants to isolate colonies resistant to 2 4 mg mL Genetici
15. for 4 hours at 37 C 5 Remove the nitrocellulose filter from the paper and replace the paper with two new sheets Soak the paper with 10 15 mL of 0 1 N NaOH 1 5 M NaCl 0 015 M sodium citrate Place the nitrocellulose filter face down on the paper and incubate for 5 minutes at room temperature Continued on next page Determining Copy Number of Multiple Integrants Continued Quantitative Dot 6 Remove the nitrocellulose filter and replace with two new 3MM sheets Soak Blot Procedure with 10 15 mL 2X SSC Place the nitrocellulose filter face down on the 3MM Continued 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 nonradioactive labeled or random primed 32P 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 B scanner if radiolabeled Southern Blot For a detailed description of this technique as applied to Pichia pastoris see Analysis 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 It is also important to understand that your strategy will be different if you use pPIC3 5K versus pAO815 to generate your
16. 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 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 Geneticin 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 Enzymol
17. 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 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 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994
18. multiple copies 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 to estimate gene dosage Controls It is very important to include DNA from the host strain alone GS115 or KM71 the host strain transformed with the parent vector pPIC3 5K or pAO815 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 one mutant and the other wild type see Recombination and Integration in Pichia Pichia Expression Kit manual 43 Accessory Products Introduction Primers Other Pichia The following products may be used with the pPIC3 5K and pAO815 vectors For details visit www invitrogen com or contact Technical Support see page 45 Item Amount Catalog no Electrocomp TOP10F 5 x 80 uL C665 55 pu Chemically 20 x 50 uL C3030 03 S N A P Gel Purification Kit 25 preps K1999 25 1 gram 11811 023 Geneticin 5 grams 11811 031 25 grams 11811 098 For your convenience Invitrogen offers a cus
19. the DNA Resuspend in 17 uL of sterile water Set up the dephosphorylation reaction in a microcentrifuge tube as follows BamH I digested recombinant pAO815 page 16 Step 2 ME 10X CIP Buffer 2 uL CIP 1 Unit uL luL Total Volume 20 uL Incubate at 37 C for 15 minutes Add 30 uL sterile water to the reaction to make a final volume of 50 uL Add 50 uL phenol chloroform and extract your DNA solution Transfer the aqueous solution to a new tube Precipitate the DNA by adding 5 uL 3 M sodium acetate and 110 uL 100 ethanol Incubate on ice for 30 minutes Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant Wash the nucleic acid pellet with 80 ethanol centrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol and air dry the pellet Resuspend the pellet in 8 uL sterile water Save on ice if you plan to ligate your insert immediately see Ligating and Digesting the Expression Cassette next page or store at 20 C Continued on next page 17 pAO815 n Vitro Multimerization Protocol Continued Ligating and Digesting the Expression Cassette 18 Ligation of the expression cassette will generate head to tail head to head and tail to tail multimers Creation of head to tail multimers will be in the correct orientation for expression and will destroy both the Barn H I and Bgl II sites between the expression cassettes Dige
20. 3 5K is used as a SCREEN not as a SELECTION for multicopy integrants Continued on next page Product Overview Continued Generating Multicopy Inserts in vitro B The graphic below shows how pAOS15 is used to generate multiple expression cassette copies in a single vector prior to transformation into Pichia The gene of interest is inserted into the vector at a unique EcoR I site The resulting expression cassette the P 45x plus your gene is flanked on the upstream side by a unique Bgl II site and on the downstream site by a unique BamH I site see A below The vector containing the gene of interest is digested with Bgl II and BamH I to excise the expression cassette The cassette is then reinserted at the BamH I site to create a tandem repeat of the cassette The reinsertion process can be repeated to generate a series of vectors that contain an increasing number of cassettes linked to a single HIS4 gene see B below Transformation of Pichia with these in vitro formed multimers increases the frequency of multicopy expression cassette recombinants Pichia recombinants may be custom designed to contain a defined number of multicopy inserts For more information see page 12 Bg Il EcoR EcoRI BamHI Vector 5 AOX1 Paox Gene of interest TT HIS4 1 Expression Cassette Digestion V 7 with BamH Recombinant Vector Bgl Il BamH la BamH Recombinant EJAS C CTAG GATC cN Vector G 1 Exp
21. 5 proceed to In Vitro Multimerization next page If you cloned your insert into pPIC3 5K generate enough plasmid DNA to transform Pichia 5 10 ug of each plasmid per each transformation Proceed to Transformation into Pichia page 23 11 pAO815 n Vitro Multimerization Protocol Introduction At this point you should have your gene cloned into the EcoR I site of pAO815 recombinant pAO815 You will use this vector for two purposes First you will use it to generate a Bgl II BamH I expression cassette consisting of the AOX1 promoter and your gene Second you will linearize the vector using BamH I to allow cloning of multiple copies of the Bg II BamH I expression cassette Note that the linearized vector already contains one copy of your expression cassette To generate multiple copies of your expression cassette Step Description 1 Treat your Bgl II BamH I expression cassette with ligase in vitro Note that Bgl II and BamH I share 4 bases in common between their recognition sites 2 Generate head to tail head to head and tail to tail multimers Head to tail ligation which is the correct orientation for expression will destroy both the BamH I and Bgl II sites 3 Treat the ligation mix with BamH I and Bg II to eliminate head to head and tail to tail multimers 4 Ligate into BamH I linearized recombinant pAO815 5 Transform into E coli and analyze recombinant plasmids for copy number by digesting wi
22. 7 primer site bases 855 875 EcoR Site bases 943 948 3 AOX7 primer site bases 1024 1044 3 AOX1 transcription termination TT bases 950 1277 HIS4 ORF bases 4199 1665 3 AOX1 fragment bases 4554 5310 pBR322 origin bases 6394 5740 Ampicillin resistance gene bases 7399 6539 35 Recipes YPD Geneticin plates 36 Yeast Extract Peptone Dextrose Medium 1 yeast extract 2 peptone 2 dextrose glucose 1 5 agar Variable amounts of Geneticin 10X D 20 Dextrose Dissolve 200 g of D glucose in 1 000 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 from Invitrogen see page 44 Prepare 30 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 Autoclave for 20 minutes on liquid cycle 3 Add 25 mL of 10X D and mix well 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 5 Mix well by swirling but be careful to minimize bubble forma
23. A ATTCCCTAGG GCGGCCGCGA ATTAATTCGC CTTAGACATG ACTGTTCCTC AGTTCAAGTT GGGCACTTAC 3 AOX 1 Primer Site 1055 1075 pLM GAGAAGACCG GTCTTGCTAG ATTCTAATCA AGAGGATGTC AGAATGCCAT m TTGCCTGAGA GATGCAGGCT TCATTTTTGA TACTTTTTTA TTTGTAACCT AOXI mRNA 3 end 1146 ATATAGTATA GGATTTTTTT TGTCATTTTG TTTCTTC Special e For pPIC3 5K the fragment containing the gene of interest should have a Considerations Kozak consensus sequence for proper translation initiation although this requirement is not as stringent in yeast For example ACC ATG G isa Kozak consensus sequence where the ATG corresponds to the initiating ATG for your gene of interest Cavener and Stuart 1991 Kozak 1987 and Kozak 1990 Note There is an ATG upstream of the SnaB I site e Be sure to analyze the 5 untranslated region of the mRNA for secondary structure formation Secondary structure in the mRNA may have a negative effect on expression of the recombinant protein e Ifyou are digesting with BamH I and SnaB I or SnaB I and EcoR I digest with SnaB I first If you digest with BamH I or EcoR I first the SnaB I site will be too close to the end of the DNA and will not be digested properly Continued on next page Cloning into pPIC3 5K and pAO815 Continued Paox1 and Multiple The sequence below shows the details of the multiple cloning site and Cloning Site of pAO815 surrounding sequences AOX1 mRNA 5 end 824 5
24. Chain Reaction Nucleic Acids Res 18 6460 Simpson R T Thoma F and Brubaker J M 1985 Chromatin Reconstituted from Tandemly repeated Cloned DNA Fragments and Core Histones A Model System for the Study of Higher order Structure Cell 42 799 808 Takeshita S Tezuka K i Takahashi M Honkawa H Matsuo A Matsuishi T and Hashimoto Gotoh T 1988 Tandem Gene Amplification in vitro for Rapid and Efficient Expression in Animal Cells Gene 71 9 18 Taylor W H and Hagerman P J 1987 A General Method for Cloning DNA Fragments in Multiple Copies Gene 53 139 144 Transformation into Pichia Introduction Linearizing Plasmid DNA N S aENo 4 O O E Nous l At this point you should have your gene cloned as multimers in pAO815 and singly in pPIC3 5K You should also have about 5 10 ug of each construct for each transformation into Pichia For methods to transform Pichia and select Hist transformants refer to the Pichia Expression System manual To linearize your construct prior to transformation into Pichia see below To linearize your construct in pPIC3 5K or pAO815 read the following 1 If you cloned your insert into pPIC3 5K you will need to linearize your insert prior to transformation using e BglII for replacement at AOX1 GS115 e SacI for insertion at AOX1 GS115 or KM71 e SalI for insertion at HIS4 GS115 or KM71 If you cloned your insert into pAO815 you will nee
25. Invitrogen by technologies pPIC3 5K pAO815 Pichia vectors for multicopy integration and intracellular expression Catalog no V173 20 V180 20 Rev Date 14 July 2010 Manual part no 25 0156 MAN0000038 ii Contents Kit Contents anid Storage seeded entes edet ene ie ori deett deine tee E ei eee hrs does v Introduction TREE 1 Product OVerview nuce eak ne E p ec dd npe np iet ob beers ree EEA S e Ed 1 MethodS 4 eroe rsen tois sent asus eus ul elo cue dke uror Me eo OSEE MIN ESCRRE TREE eDLEe DR MEAE RECEN 6 Cloning into pPIC3 5K and PAOS Ia aopean ee e i n aaan eSEE EEO AAS SRA EEES tenentes 6 Analyzing E coli Transformants saisie nine ee oia ite ns ries dee i decetero ine i bran 11 pAOS15 In Vitro Multimerization Protocol sse 12 Transformation into Pichia ii iei e Ripe ee 23 pPIC3 5K In Vivo Screening of Multiple Inserts sse 27 AppendD6nuii n uaiaiseiied ro eaaa era aa Iu UI SEI DLYNRMA INI I IU aaa e danaa daana ianh 33 bier c OU NERA 33 PRICI JK suenie niea M M 34 1 SYN G ofl io uade Od nU Mns S LA aUe a e UAE ot a etel itta Dod Ra ADM ese Era 35 Recipes aont oo d bae v cadendo indietro E deayee e botte E at 36 Pichia Genomic DNA Isolation ssssssssssssseeeeeeeeene nennen tenete tenente 37 Easy DNA Protocol for Isolation of DNA from Pichia eese tete 39 Determining Copy Number of Multiple Integrants ssssss
26. Manual Sambrook et al 1989 e EcoRI BamH I and Bgl II restriction enzymes and appropriate buffers e Agarose and low melt agarose e S N A P Gel Purification Kit see page 44 or glass milk e Sterile water e CIP calf intestinal phosphatase 1 unit WL e 10X CIP Buffer e Phenol chloroform e 3Msodium acetate e 100 ethanol e 80 ethanol e T4 Ligase 2 5 units uL e 10X Ligation Buffer with ATP e LB medium e LB ampicillin plates 50 100 ng mL ampicillin e 16 C 37 C and 65 C water baths or temperature blocks e Geneticin antibiotic see page 44 e YPD Geneticin plates see Recipes page 36 e 50 mL conical centrifuge tubes e Hemacytometer e 30 C and 37 C incubator e Microtiter plates optional Registered Pichia users should already have the Pichia Expression System and the current manual Procedures for transformation into E coli and Pichia analysis of recombinants and expression are described in the Pichia manual Introduction Product Overview Description of the System Frequency of Multicopy Inserts Generating Multicopy Inserts in vivo 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 two vectors include
27. References page 47 e We recommend that you ligate your insert into both pPIC3 5K and pAO815 so that you can try both methods to isolate multiple integrants Below are some guidelines to consider when developing a cloning strategy for these vectors The multiple cloning sites for each vector are presented on the pages 9 10 for your convenience e Werecommend that you transform the two supercoiled Pichia expression vectors into E coli so that you have a permanent stock and a way to make more plasmid Transform competent E coli with 1 2 uL of the supplied vector stock solution and select on LB with 50 100 ng mL ampicillin LB Amp The following general considerations are applicable to both vectors 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 Maintain plasmid constructions in a recA endA mutant E coli strain such as TOP10 Electrocompetent TOP10 cells are available from Invitrogen see page 44 e The native 5 end of the AOX1 mRNA is noted in each multiple cloning site 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 each figure on pages 9 10 e The premature te
28. S115 and Hist Mut in KM71 For insertion at HIS4 linearize with Sal I generates Hist Mut in GS115 and His Mut in KM71 For a gene replacement at AOX1 in GS115 linearize with Bg II generates Hist Mut See page 24 for alternate restriction sites if your insert DNA has a Bgl II Sac I or Sal I site pAOS15 is a plasmid designed for in vitro generation of multimers of your gene for integration into the Pichia genome 7709 bp vector One unique restriction site EcoR I Intracellular expression of your gene Requires an initiating ATG codon in a Kozak consensus sequence for proper translation initiation of your gene Cavener and Stuart 1991 Kozak 1987 Kozak 1990 HIS4 selection in Pichia For insertion at HIS4 linearize with Sal I or Stu I generates Hist Mutt in GS115 and His Mut in KM71 For a gene replacement at AOX1 in GS115 linearize with Bg II generates His Mut5 See page 24 for alternate restriction sites if your insert DNA has a Bgl IL Stu I or Sal I site Methods Cloning into pPIC3 5K and pAO815 General Molecular Biology Techniques iJ MEND 7 1 RECO General Considerations For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 See
29. ay result in the isolation of false positives In vitro Method pAO815 Advantages Quantitative construction of a defined number of multimers Most of the His transformants will contain the proper defined number of inserts Isolation of recombinants with multiple inserts is easier because most of the His transformants contain multiple copies of your gene In vitro construction allows step wise analysis of copy number effects on protein expression Multiple inserts are located at a single locus No need for a second drug resistance marker in the vector Disadvantages More work up front to clone defined number of multimers Size of the vector may become quite large depending on the size of your gene and the number of copies you create Rearrangements in E coli may occur Continued on next page Product Overview Continued pPIC3 5K pAO815 pPIC3 5K is a plasmid designed to allow you to identify in vivo multiple integrations of your gene in the Pichia genome 9004 bp vector Five unique restriction sites in the multiple cloning site BamH I SnaB I EcoR I Avr II Not I Intracellular expression of your gene Requires an initiating ATG codon in a Kozak consensus sequence for proper translation initiation of your gene Cavener and Stuart 1991 Kozak 1987 Kozak 1990 HIS4 selection in Pichia For insertion at AOX1 in GS115 or KM71 linearize with Sac I generates His Mut in G
30. ca 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 75Mammonium 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 OD 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 Add2mL of 176 SDS mix gently and set on ice for 5 minutes 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 37 Pichia Genomic DNA Isolation Continued Precipitating DNA 38 Transfer the supernatant from Step 5 previous page and add 2 volumes of ethanol to the supernatant Incubate at room temperature for 15 minutes Centrifuge at 10 000 x g for 20 minutes at 4 C Resuspend the pellet gently in 0 7 mL 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
31. cin gene for in vivo screening of multiple copy inserts It is identical to pPIC3 5 except for the presence of the kanamycin gene pAO815 is similar to pHIL D2 except that it does not contain an f1 origin Features The table below describes the general features of the pPIC3 5K and pAO815 Pichia expression vectors Feature Description Benefit 5 AOX1 An 1 000 bp fragment containing the Allows methanol inducible high level AOX1 promoter expression in Pichia Targets plasmid integration to the AOX1 locus MCS Multiple Cloning Site Allows insertion of your gene into the expression vector TT Native transcription termination and Permits efficient transcription termination and polyadenylation signal from AOX1 gene polyadenylation of the mRNA 260 bp HIS4 Pichia wild type gene coding for Provides a selectable marker to isolate Pichia histidinol dehydrogenase 2 4 kb and recombinant strains used to complement Pichia his4 strains 3 AOX1 Sequences from the AOXT gene that are Targets plasmid integration at the AOX1 gene further 3 to the TT sequences 650 bp Amp Ampicillin resistance gene Allows selection replication and maintenance pBR322 origin E coli origin of replication in E coli BamH I Unique restriction sites Permits linearization of vector for efficient Bel II Note Stu I is not unique to pPIC3 5K integration into the Pichia genome and Not I generation of either Mut or Mut
32. d cells in 1 mL fresh 1 M Sorbitol 100 mM EDTA 14 mM B mercaptoethanol Vortex to resuspend Add 1 5 uL of 3 mg mL Zymolyase to each tube of cells and incubate at 30 C for 1 hour Centrifuge at 1 700 x g in a 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 Gently resuspend the cells in 200 uL fresh SCED and incubate at 37 C for 1 hour Continued on next page 39 Easy DNA Protocol for Isolation of DNA from Pichia Continued Isolating DNA 40 god Gon he 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 8076 ethanol centrifuge at maximum speed for 2 minutes at 4 C remove ethanol and air dry 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 uL PCR reaction Determining Copy Number of Multiple Integrants General Guidelines
33. d in this kit allow isolation and generation of multicopy inserts by in vivo or in vitro methods to test whether increasing the copy number of your recombinant gene will lead to a subsequent increase in protein expression The in vivo method utilizes hyper resistance to Geneticin G418 sulfate to screen for possible multicopy inserts while the in vitro method produces tandem inserts of your gene by ligation 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 The in vitro method allows you to construct multimers by ligation When His transformants are selected they have a high probability of containing the multimers that you constructed in vitro pPIC3 5K 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 pPIC3 5K integrated into the Pichia genome confers resistance to Geneticin to a level of 0 25 mg mL Multiple integrated copies of pPIC3 5K can increase the Geneticin resistance level from 0 5 mg mL 1 2 copies up to 4 mg mL 7 12 copies Because of the genetic linkage between the kanamycin gene and the expression cassette
34. d to linearize your insert prior to transformation using e Bgl II for replacement at AOX1 GS115 e Sallor Stu for insertion at HIS4 GS115 or KM71 Note that multiple Sac I sites are formed if there are 2 or more multimers in pAO815 If your insert contains any of these restriction sites see the table on the next page for alternate sites We recommend 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 If you want only Mutt recombinants Linearize pPIC3 5K with Sac I or Sal I for insertion at AOXT or his4 respectively and transform GS115 Linearize pAO815 with SalI or Stu I for insertion at his4 and transform GS115 If you wish to have only Mut recombinants Use strain KM71 which is already Mut and linearize for insertion at AOX1 or his4 Linearize pPIC3 5K with Bgl II for gene replacement at AOX1 and transform GS115 Linearize pAO815 with Bgl II for gene replacement at AOX1 and transform GS115 Continued on next page 23 Transformation into Pichia Continued Alternate Restriction Sites 24 The table below describes alternate restriction sites for linearizing your construct before transformation into Pichia pPIC3 5K Note that an additional Stu I site was added with the inclusion of the kan gene so that the Stu I site in HIS4 is no longer unique
35. ding 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 the Company s liability to only the price 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 The Company reserves the right to select the method s used to analyze a product unless the Company 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 the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have 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 45 Purchaser Notification Limited Use Label License No 74 Pichia Pastoris Expression System 46 The Pichia Expression System is based on the yeast Pichia pastoris
36. dium citrate e 2X SSC 1X 0 15 M NaCl 0 015 M sodium citrate e 3MM paper The following protocol is a summary of a rapid DNA dot blot technique to detect multiple integrants Romanos et al 1991 It is very important to spot equivalent numbers of cells onto filters to quantify copy number 1 Grow Mutt or Muf transformants in individual wells of a 96 well microtiter plate in 200 uL of YPD broth at 30 C until all wells have approximately the same density This may necessitate several passages see page 27 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 multichannel pipettor Air dry filters 3 Tolyse the cells on the filter 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 B mercaptoethanol pH 9 0 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 the paper 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
37. ecombinant vector Construct is unstable in Use the in vivo method to isolate rearranges and deletions are E coli multimers see page 27 detected Pichia His transformants do Vector was linearized with Linearize your construct with Sal I or not have multimers the wrong enzyme Stu I to insert the construct into his4 on ENZYMES m the Analyze your construct for other 2 AOAI eon ATE unique restriction sites in the vector duplicated when backbone that are near the 5 AOX1 multimers are created region or the 3 AOX1 region These sites will preserve your multimers and allow recombination with AOXI Continued on next page 21 pAO815 n Vitro Multimerization Protocol Continued For More Information 22 There are a number references in the literature you can consult to optimize synthesis of in vitro multimers A partial list is provided below Cohen B and Carmichael G G 1986 A Method for Constructing Multiple Tandem Repeats of Specific DNA Fragments DNA 5 339 343 Eisenberg S Francesconi S C Civalier C and Walker S S 1990 Purification of DNA Binding Proteins by Site specific DNA Affinity Chromatography Methods Enzymol 182 521 529 Graham G J and Maio J J 1992 A Rapid and Reliable Method to Create Tandem Arrays of Short DNA Sequences BioTechniques 13 780 789 Rudert W A and Trucco M 1990 DNA Polymers of Protein Binding Sequences Generated by Polymerase
38. ector backbone Analyze your digests on a 17o agarose gel You should see bands corresponding to 1 copy 2 copies 3 copies etc of your expression cassette along with the vector backbone The number of copies you obtain may depend on how well a large vector is tolerated by the host strain Once you have identified plasmids with multiple copies of your expression cassette be sure to purify by streaking for single colonies and confirming your construct Prepare frozen glycerol stocks of E coli containing each of your multimeric constructs Prepare 5 10 ug of each plasmid for transformation into Pichia Proceed to Transformation into Pichia page 23 Continued on next page pAO815 n Vitro Multimerization Protocol Continued Troubleshooting The table below will help you optimize formation and isolation of multimers in Pichia Problem Possible Reason Solution No multimers or low CIP defective Use fresh CIP number of multimers in Add more CIP Add 1 unit of CIP and your vector after transformation into E coli incubate 15 more minutes at 37 C This is somewhat risky as CIP can degrade the ends of your DNA Not enough insert DNA to Digest more pAO815 containing ligate 1 copy of your expression cassette Construct is unstable in Use the in vivo method to isolate E coli multimers see page 27 Multimers are too long to Try ligating each expression cassette ligate efficiently separately R
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40. ernative Procedure Materials Needed You may wish to build each desired multimer in increments by ligating each additional expression cassette one or two at a time into pAO815 For example Step Description 1 Digest pAO815 with one copy of your gene using BamH I 2 Ligate a single copy of the Bg II BamH I expression cassette into the vector 3 Transform E coli and analyze the transformants for the vector with 2 copies of your insert 4 Isolate and digest this vector with 2 copies of your gene with BamH I and Bg II to isolate a cassette with 2 copies of your gene optional 5 Digest the vector with 2 copies of your gene with BamH I and ligate 1 or 2 copies see Step 4 of the expression cassette into the vector 6 Transform E coli and analyze the transformants for the vector with 3 or 4 copies of your insert 7 Repeat until the desired multimer is reached Electrocompetent or chemically competent E coli must be recA end A for transformation You will need 3 4 tubes of competent cells per experiment See page 44 for ordering information EcoR I BamH I and Bgl II restriction enzymes and appropriate buffers Low melt agarose S N A P Gel Purification Kit see page 44 or glass milk Sterile water CIP calf intestinal alkaline phosphatase 1 unit uL 10X CIP Buffer Phenol chloroform 3 M sodium acetate 100 ethanol 80 ethanol T4 Ligase 2 5 units uL 10X Ligation
41. he amount you to transform into electrocompetent cells to prevent arcing Remember to include the vector only and cells only controls to evaluate your experiment The vector only will indicate whether your vector was dephosphorylated Since the CIP reaction is not 100 and because you often get degradation of the ends there might be a few colonies on this plate The cells only plate should have no colonies at all 1 Transform competent E coli by your method of choice 2 After adding medium to the transformed cells and allowing them to recover plate 10 uL and 100 uL of each transformation mix onto LB plates with 50 100 ug mL ampicillin Save the remainder of your transformation mix at 4 C 3 Incubate overnight at 37 C If you do not get transformants or very few transformants plate out the remainder of the transformation mix onto LB ampicillin plates Continued on next page 19 pAO815 n Vitro Multimerization Protocol Continued Analyzing Transformants 20 Pick 20 transformants and inoculate 2 mL LB containing 50 100 pg mL ampicillin Grow overnight at 37 C Isolate plasmid DNA and digest with Bg II and BamH I to release any multimers from pAO815 Be sure to include Bgl II BamH I digested pAO815 as a control It is possible to get vector rearrangements and deletions with large vectors in E coli Including Bg II BamH I digested pAO815 will allow you to detect these rearrangements deletions in the v
42. lonies at a later date Continued on next page 29 pPIC3 5K n Vivo Screening of Multiple Inserts Continued Method 2 30 You will need three sets of two microtiter plates 6 total to screen 180 Hist recombinants It is important to 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 re plate them on minus histidine plates see Pichia Expression System manual 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 microtiter plates and add 190 uL of YPD to each well 5 Inoculate the second set of microtiter plates with 10 uL from the first set of microtiter plates by using a multichannel pipette Make sure the second set of plates is marked and oriented in such a way that you can keep track of wells Cover and incubate the second set of plates overnight at 30 C The next
43. ls such as TOP10 see page 44 for ordering using your method of choice Analyzing E coli Transformants Analyzing Transformants Sequencing Recombinant Clones After Sequencing After transformation plate 10 uL and 100 uL of the transformation mix onto LB plates with 50 100 ug mL ampicillin and select ampicillin resistant colonies Pick 10 ampicillin resistant transformants and inoculate into 2 mL LB medium with 50 100 ug mL ampicillin Grow overnight at 37 C with shaking Isolate plasmid DNA by miniprep for restriction analysis and sequencing see below To sequence the Pichia expression vectors pPIC3 5K and pAO815 with only one insert use the 5 AOXI and the 3 AOX1 sequencing primers Make a glycerol stock of your desired clone 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 80 C We strongly recommend that you sequence your construct in pPIC3 5K and pAO815 with only one insert before proceeding further to confirm that the ATG is in the proper context for eukaryotic translation initiation Use the 5 and 3 AOXI sequencing primers to sequence your constructs Sequencing primers are available from Invitrogen see page 44 Once you have cloned and sequenced your insert proceed as directed below If you cloned your insert into pAO81
44. n 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 one copy construct controls to analyze your PCR experiment Since there is no guarantee that multiple copies will actually increase the amount of protein expressed you may elect to proceed directly to expression to see if any of these colonies overexpress 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 your clones by streaking for single colonies and making frozen glycerol stocks of all your Geneticin resistant colonies Always initiate expression studies from frozen stocks not old plates If you find that your Geneticin resistant His recombinants significantly overexpress 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 page 41 It is very important to include genomic DNA isolated from Pichia recombinants transformed with pPIC3 5K alone and pPIC3 5K
45. nual Analyze for the presence of your insert by PCR see the Pichia Expression System manual for a protocol Note The size of the PCR product for pAO815 is 189 bp PCR will only indicate if your gene is present but will not indicate how many copies of your gene are integrated or at which locus it is integrated PCR can reasonably be done on 12 20 transformants Remember to include vector only and original construct controls 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 overexpress their protein Be sure to include a single copy insert as a control Test all your multimeric Hist transformants for their Mut phenotype so that you induce expression properly Refer to the Pichia Expression System manual for methods to express your protein Continued on next page 25 Transformation into Pichia Continued Determining Copy If you find that your His recombinants significantly overexpress your protein Number you may wish to quantify the copy number of your gene Copy number may be analyzed by Southern or quantitative dot slot blots see page 41 It is very important to include genomic DNA isolated from the host strain Pichia recombinants transformed with the parent vector and Pichia recombinants transformed with pPIC3 5K or pAOS815 containing a single copy of you
46. ogy 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 Continued on next page 47 References Continued 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 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 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective Owners 48 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrog
47. r gene as controls to evaluate your experiment X 17 Be sure to purify your clones by streaking for single colonies and making frozen MEN D L TENES E x ore SV ND glycerol stocks of all your Geneticin resistant colonies Always initiate E E expression studies from frozen stocks not old plates P 26 pPIC3 5K n Vivo Screening of Multiple Inserts Introduction You will need as many His transformants as you can conveniently generate Note 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 Methods to Screen There are two methods used to screen His transformants for Geneticin for Geneticin resistance Resistant e Method 1 is technically easier and screens a greater number of clones but is Transformants less reliable After initial selection of His transformants they are pooled and plated on YPD Geneticin plates containing increasing concentrations of Geneticin Method 1 is applicable to spheroplast or electroporation transformation methods e Method 2 is technically more difficult and screens fewer numbers of clones
48. recombinants SacI Sal I StuI kan Kanamycin resistance gene from Tn903 Allows in vivo screening for multicopy inserts by which confers resistance to Geneticin in increased resistance to Geneticin Pichia and kanamycin resistance in F coli Also allows selection for kanamycin resistance for pPIC3 5K only in E coli There is no yeast origin of replication in any of the Pichia expression vectors Note available from Invitrogen Hist transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome i e integration of the plasmid 33 pPIC3 5K Map of pPIC3 5K The figure below shows the map of pPIC3 5K Details of the multiple cloning site are shown on page 9 3 AOX1 TT Comments for pPIC3K 9004 nucleotides 5 AOX1 promoter fragment bases 1 937 T Sal 5 AOX1 primer site bases 855 875 g Multiple Cloning Site bases 938 968 3 AOX1 primer site bases 1055 1075 3 AOX7 transcription termination TT bases 981 1314 HIS4 ORF bases 4242 1708 Kanamycin resistance gene bases 5458 4656 BspE 3 AOX1 fragment bases 5850 6607 pBR322 origin bases 7689 7016 Ampicillin resistance gene bases 8694 7834 34 pAO815 Map of pAO815 The figure below shows the map of pAOS15 Details of the multiple cloning site are shown on page 10 nd Q o LU 3 AOX1 TT BamH Comments for pAO815 7709 nucleotides 5 AOX1 promoter fragment bases 1 940 5 AOX
49. ression j Cassette Bgl I BamH la Insert GATC T IR ETRA C CTAG A Bgl Il BamH I Bgl II BamHI 5 AOX1 Paox Gene of interest TT 5 AOX1 Paox Gene of interest TT HIS4 2 Expression Cassettes Continued on next page Product Overview Continued We recommend trying both methods to generate or isolate multicopy inserts of your gene A summary of the advantages and disadvantages of each method is presented in the lists 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 your protein In vivo Method pPIC3 5K Advantages Easier to initiate experiment because only e one copy of your gene is cloned into pPIC3 5K before transforming into Pichia Identifies the 1 10 of spontaneous His e transformants that have multiple inserts Average size of vector is similar to other e Pichia expression vectors Multiple inserts are located at a single locus Disadvantages Qualitative screen Geneticin resistance may not necessarily correlate with the number of copies of your gene Screening His transformants may involve more work because you need thousands of His transformants to generate enough Geneticin resistant colonies to test The number of multiple inserts is unknown although this can be determined through Southern or dot blot analysis Screening on Geneticin is sensitive to the density of the cells and m
50. rmination of transcripts because of AT rich regions has been observed 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 to express your gene Scorer et al 1993 Continued on next page Cloning into pPIC3 5K and pAO815 Continued General Cloning Strategies generally fall into three different categories Strategies 1 Ligation of a compatible restriction fragment a Forced directional insertion involving the use of two different sites in the multiple cloning site pPIC3 5K b Ligation of the fragment with the same restriction end on both ends into a single compatible site e g EcoR I cloning in pAO815 Note that you will need to dephosphorylate pAO815 to ligate into the EcoR I site 2 PCRamplification 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 see page 44 followed by subcloning of a compatible fragment into the appropriate Pichia expression vector If your insert has an EcoR I site and you are trying to clone into the EcoR I site of Important pAO815 we recommend the following 1 An en
51. rmine 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 uL 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 Plate 105 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 the YPD plates without Geneticin 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 9 106 and 107 dilutions of the pooled transformants using sterile water Plate 100 to 200 uL per plate Incubate plates at 30 C and check daily Geneticin resistant colonies will take 2 to 5 days to appear while cells plated on YPD without Geneticin will take 2 3 days Proceed to Analyzing the Results page 31 Continued on next page pPIC3 5K n Vivo Screening of Multiple Inserts Continued Method 1 Use this procedure if electroporation was used to transform Pichia Electroporation Transformants will not be plated in top agar Start with plates containing His transformants 1 Pipette 1 to 2 mL sterile water over the
52. s containing your gene These will produce a band that will be quite intense The relative intensity of this band versus a band containing a single copy of your gene will allow you to determine the copy number Bel II digested DNA from GS115 and GS115 transformed with pPIC3 5K or pAO815 will produce a bands of 2 8 kb the genomic copy of HIS4 and 6 7 kb the vector derived copy of HIS4 respectively when probed with a complementary fragment to HIS4 41 Determining Copy Number of Multiple Integrants Continued Introduction Quantitative Dot Blot Solutions Quantitative Dot Blot Procedure 42 You may wish to determine the actual number of gene copies in your Pichia recombinant You may 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 pAO815 or pPIC3 5K 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 37 and 39 to isolate genomic DNA You will need the following solutions 10 15 mL of each for each dot blot e 50mM EDTA 2 5 B mercaptoethanol pH 9 0 e 1mg mL Zymolyase 100T in water Seikagaku America Inc e 0 1 N NaOH 1 5 M NaCl 0 015 M so
53. s or Southern analysis e pAO815 or pPIC3 5K containing one copy of your expression cassette Be sure to linearize pAOS815 in the same manner as your multimer Most of the His transformants created by transforming with recombinant pPIC3 5K will only have one copy Make sure that the transformant you pick is only resistant to 0 25 mg mL Geneticin The single copy controls created using pPIC3 5K and pAO815 should have the same Mut phenotype as the putative multimeric recombinants you are testing These recombinants will be used as a control to compare expression levels with multiple copies of your expression cassette and as a single copy control for quantitative dot blot or Southern analysis 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 and selection of His recombinants Invitrogen also offers the EasyComp Kit for preparation and transformation of competent Pichia cells see page 44 Once you have generated His transformants using recombinant pPIC3 5K proceed to In Vivo Selection of Multiple Inserts page 27 For His transformants generated using recombinant pAO815 you will need to analyze recombinants for the presence of your insert Refer to PCR Analysis of Pichia Integrants in the Pichia Expression System ma
54. sssseseseeeeeeneenenenee 41 Accessory Products 2 cs le ex te de OR REOR Ede a ptc ee BST coat 44 Technical S pport ec due Sete dierap ee RD Dep e ENIM REGERE IRE 45 Purchaser Notifications asian odo igeta god uid ndi dade ba acd i Rp aud 46 References c aeinn ted C e I HERE ace IR SERI a a Y E Re Seca E re haie RE e dE PERDE 47 iii iv Kit Contents and Storage Shipping and pPIC3 5K and pAO815 vectors are shipped on wet ice Upon receipt store vectors Storage at 20 C Kit Contents All vectors are supplied as detailed below Store the vectors at 20 C Catalog no Vector Composition Amount gt 40 uL of 0 5 ug uL vector in 10 mM Tris HCI V173 20 pPIC3 5K 1 mM EDTA pH 8 0 20 ug f 40 uL of 0 5 ug uL vector in 10 mM Tris HCI V180 20 pAO815 1 mM EDTA pH 8 0 20 ug Intended Use For research use only Not intended for any animal or human therapeutic or diagnostic use Kit Contents and Storage Continued Materials Supplied by the User vi Important For the procedures described in this manual you will need e Manual from the Pichia Expression System e Microbiological equipment e Hlectrocompetent or chemically competent E coli must be recA end A for transformation page 44 You 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
55. stion of the multimers with BamH I and Bgl II will eliminate those multimers with tail to tail and head to head orientation After digestion with these two restriction enzymes you will have a mixture of multimers containing 1 2 3 etc copies of your gene that can be ligated into BamH I linearized recombinant pAO815 1 10 Set up a 20 uL ligation reactions as follows Bgl II BamH I digested expression cassette 15 uL Sterile water 2 uL 10X Ligation Buffer with ATP 2uL T4 DNA Ligase 2 5 units WL 1 pL Total Volume 20 uL Incubate at 16 C for 2 5 hours Heat inactivate the ligase by incubating at 65 C for 20 minutes Add the following reagents for restriction enzyme digestion cut back Note that BamH I and Bel II may be used with the same restriction buffer Sterile water 23 pL 10X restriction enzyme buffer 5 uL Bgl II 10 units mL 1uL BamH I 10 units mL 1 pL Total Volume 30 uL Incubate the reaction at 37 C for 2 hours Add 50 uL phenol chloroform and extract the restriction enzyme digestion to remove the enzymes Transfer the aqueous solution to a new microcentrifuge tube To ethanol precipitate the DNA add 5 uL 3 M sodium acetate and 110 uL 100 ethanol Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant Wash the nucleic acid pellet with 80 ethanol centrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol
56. t 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 Expression 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
57. th Bel II and BamH I Continued on next page 12 pAO815 n Vitro Multimerization Protocol Continued Flow Chart of The figure below and on the following page outlines the multimerization Multimerization process Process Digest with Recombinant BamH Bgl ll Digest with BamH l Bgl Il Bgl M BamHI PO 1 Paox Gene of Interest TT PO Expression Cassette Treat w Ligase Bg Il BamH l Bgl II BamH PO Expression Cassette Expression Cassette PO head to tail Bgl Il BamH Bgl Il PO Expression Cassette Expression Cassette PO tail to tail BamHI Bgl Il BamHI PO Expression Cassette i Expression Cassette PO head to head Digest w BamH and Bg Il Bgl Il BamH l Bgl II BamH PO Expression Cassette Expression Cassette PO Bgl II BamHI BamHI Bgl II PO Expression Cassette Expression Cassette PO BamHI Bgl II Bgl II BamHI PO Expression Cassette Expression Cassette PO Site is not cleavable by BamH or Bg II Continued on next page 13 pAO815 n Vitro Multimerization Protocol Continued Flow Chart of Multimerization Process Continued 14 BamHI Bgl M BamHI HIS4 3 AOX1 5 AOX1 Prox TT PO Dephosphorylate the Vector BamHI Bgl BamHI HIS4 3 AOX1 5 TON TT Amp Gene of Interest Amp Gene of Interest Mix Vector and Inserts Ligate Continued on next page pAO815 n Vitro Multimerization Protocol Continued Alt
58. tion 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 Preparation Spheroplasting and Lysis The protocol below allows you to isolate DNA from the desired His 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 see page 44 to isolate DNA from Pichia for PCR and quantitative dot slot blots See page 41 for this protocol Lastly there is a fast DNA isolation protocol for multiple samples 24 which has been reported Wach et al 1994 You need to prepare the following solutions e Minimal Medium MD MGY e Sterile water e Fresh 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 Ameri
59. tom primer synthesis service Visit www invitrogen com for more details Other Pichia products available from Invitrogen are described below Products Item Purpose Amount Catalog no Pichia Expression Kit Complete Kit for Gene Expression in Pichia 1 kit K1710 01 pastoris Spheroplast Kit Preparation of Pichia spheroplasts 1 kit K1720 01 Pichia EasyComp Rapid preparation and transformation of 1 kit K1730 01 Transformation Kit competent P pastoris cells Easy DNA Kit Isolation of DNA from Pichia for PCR 1 kit K1800 01 pPICZ A B amp C For simple selection on Zeocin and 20 ug each V190 20 intracellular expression of recombinant proteins containing a C terminal histidine tag pPICZa A B amp C For simple selection on Zeocin and secreted 20 ug each V195 20 expression of recombinant proteins containing a C terminal histidine tag pPIC9K For in vivo isolation of multiple copy inserts 20 ug V175 20 for secreted expression 44 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs 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
60. with a single copy of your gene as controls to evaluate your experiment Continued on next page 31 pPIC3 5K n Vivo Screening of Multiple Inserts Continued Troubleshooting 32 Since there is a tendency to isolate false positives colonies which appear to be Geneticin resistant but are not it is very important to purify your putative Geneticin resistant colonies and confirm the observed level of Geneticin resistance before proceeding further The other most common problem with the in vivo method is that very few Geneticin resistant colonies are isolated requiring screening of more His transformants Remember that you are isolating spontaneous multiple integration events These occur at a frequency of 1 10 which may mean that you need to screen thousands of His transformants as opposed to hundreds In addition to isolate recombinants with the most copies of your gene inserted you must screen more His transformants Successive multiple insertions are simply more rare If you find that your transformation efficiency is low try electroporation instead of spheroplasting This may increase the transformation efficiency and help you isolate more His transformants Vectors Introduction Appendix The vectors pPIC3 5K and pAO815 share many of the same features see below Both are functional in Pichia strains GS115 and KM71 However pPIC3 5K has a more extensive multiple cloning site and contains the kanamy
61. zyme like Bsa I has the following restriction recognition site 5 GGTCTCN 3 CCAGAGNNNNN 2 An EcoRI site may be engineered into the recognition site for Bsa I 5 GGTCTCG AATTC 3 CCAGAGCTTAA G 3 This sequence may be added to your DNA fragment by integrating it into your PCR primer or created in vitro as an adaptor to another restriction site 4 Digest your PCR or adapted ligation product with Bsa I This will generate EcoR I overhangs on both ends of your fragment without digesting with EcoR I 5 AATTC 5 Ligate into dephosphorylated pAO815 Other enzymes that may be used are BsmA I or BsmB I Continued on next page Cloning into pPIC3 5K and pAO815 Continued Cloning Refer to Ausubel et al 1990 pages 3 16 1 3 17 3 or Sambrook et al 1989 Procedures pages 5 10 5 13 for help with cloning Bacterial Once you have decided on a cloning strategy you can use electrocompetent or Transformation chemically competent E coli for transformation Continued on next page Cloning into pPIC3 5K and pAO815 Continued Paox1 and Multiple The sequence below shows the details of the multiple cloning site and Cloning Site of surrounding sequences pPIC3 5K AOX LORNAS End 5 AOX 1 Primer Site 855 875 O TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC a AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA PORE map SEMI aid I Nor I CAACTAATTA TTCGAAGGAT CCTACGTAG

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