pBAD Directional TOPO® Expression Kit
Contents
1. Assay for expression of your protein of interest 19 21 Methods Designing PCR Primers Designing Your PCR Primers Specific Features for Expression Guidelines to Design the Forward PCR Primer Example of Forward Primer Design Note The design of the PCR primers to amplify your gene of interest is critical for expression Consider the following when designing your PCR primers e Sequences required to facilitate directional cloning see below e Whether or not you wish to clone your PCR product in frame with the C terminal V5 epitope and polyhistidine region The pBAD202 D TOPO vector contains the following features to facilitate expression e Initiation ATG that is properly spaced from the optimized ribosome binding site to ensure optimal translation e HP thioredoxin which acts as a translation leader to facilitate high level expression and in some cases solubility HP thioredoxin can be removed TM after protein purification using enterokinase e g EKMax see page 34 When designing your forward PCR primer consider the following points below Refer to page 8 for a diagram of the TOPO Cloning site for the pBAD202 D TOPO vector e To enable directional cloning the forward PCR primer must contain the sequence CACC at the 5 end of the primer see Example below The 4 nucleotides CACC base pair with the overhang sequence GTGG in the pBAD202 D TOPO vector e I2. The PureLink Quick Gel Extraction Kit allows you to rapidly purify PCR products from regular agarose gels see page 34 for ordering information 1 Equilibrate a water bath or heat block to 50 C 2 Cutthe area of the gel containing the desired DNA fragment using a clean sharp blade Minimize the amount of surrounding agarose excised with the fragment Weigh the gel slice 3 Add Gel Solubilization Buffer GS1 supplied in the kit as follows e For lt 2 agarose gels place up to 400 mg gel into a sterile 1 5 mL polypropylene tube Divide gel slices exceeding 400 mg among additional tubes Add 30 uL Gel Solubilization Buffer GS1 for every 10 mg of gel e For gt 2 agarose gels use sterile 5 mL polypropylene tubes and add 60 uL Gel Solubilization Buffer GS1 for every 10 mg of gel 4 Incubate the tube at 50 C for 15 minutes Mix every 3 minutes to ensure gel dissolution After gel slice appears dissolved incubate for an additional 5 minutes 5 Preheat an aliquot of TE Buffer TE to 65 70 C 6 Place a Quick Gel Extraction Column into a Wash Tube Pipette the mixture from Step 4 above onto the column Use 1 column per 400 mg agarose 7 Centrifuge at 212 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube 8 Optional Add 500 uL Gel Solubilization Buffer GS1 to the column Incubate at room temperature for 1 minute Centrifuge at 212 000 x g for 1 minute Discard the flow thro
3. To ensure low levels of expression you may find it useful to utilize glucose or glycerol to further repress the araBAD promoter Follow the steps below to express your protein 1 Transform your construct into LMG194 LMG194 can be grown in RM medium that enables repression of araBAD promoter by glucose or glycerol see Using LMG194 page 17 2 Follow the Pilot Expression protocol page 18 using RM medium containing 0 2 glucose or glycerol see page 25 for recipe to grow the cells 3 Monitor the OD oo because the cells grow more slowly in RM medium Induce with various concentrations of arabinose as described in the Pilot Expression protocol 5 Monitor ODeo over time to make sure that the cells are growing TM You may use the ProBond Purification System the Ni NTA Purification System or a similar product to purify your 6xHis tagged protein see page 35 for ordering information Both purification systems contain a metal chelating resin specifically designed to purify 6xHis tagged proteins Before starting consult the ProBond or Ni NTA Purification System manual to familiarize yourself with the buffers and the binding and elution conditions If you are using another resin follow the manufacturer s instructions The enterokinase EK recognition site can be used to remove the N terminal leader from your recombinant fusion protein after purification Note that after digestion with enterokinase there will be sev
4. Cloning to directionally clone a blunt end PCR product into a vector for soluble regulated expression and simplified protein purification in E coli Blunt end PCR products clone directionally at greater than 90 efficiency with no ligase post PCR procedures or restriction enzymes required In addition pBAD202 D TOPO vector contains the His Patch HP thioredoxin leader for increased translation efficiency and solubility of recombinant fusion proteins Expression in E coli is driven by the araBAD promoter Psap The AraC gene product encoded on the pBAD202 D TOPO vector positively regulates this promoter pBAD202 D TOPO is designed to facilitate rapid directional TOPO Cloning of blunt end PCR products for regulated expression in E coli Features of the vector include e araBAD promoter Paap for tight dose dependent regulation of heterologous gene expression e N terminal His Patch thioredoxin for increased translation efficiency and solubility of heterologous proteins e Directional TOPO Cloning site for rapid and efficient directional cloning of a blunt end PCR product see next page for more information e C terminal fusion tag for detection and purification of recombinant fusion proteins e Kanamycin resistance gene for selection in E coli e araC gene encoding a regulatory protein for tight regulation of the Paap promoter e pUC origin for maintenance in E coli Note Although the pBAD202 D TOPO vector
5. Materials Required Pilot Expression SOB or LB containing 50 pg mL kanamycin 37 C shaking incubator 20 L arabinose provided Additional L arabinose is available from Sigma Cat no A3256 In addition to testing your transformants we recommend that you include pBAD202 D lacZ as a positive control and cells without vector as a negative control 1 For each transformant or control inoculate 2 mL of SOB or LB containing 50 pg mL kanamycin with a single recombinant E coli colony Note If you are using LMG194 as a host use RM medium containing glucose and 100 pg mL ampicillin for overnight growth see page 25 for a recipe and then substitute glycerol for glucose in medium at Step 3 below see Using LMG194 previous page Grow overnight at 37 C with shaking 225 250 rpm to ODeoo 1 2 The next day label five tubes 1 through 5 and add 10 mL of SOB or LB containing 50 pg mL kanamycin Inoculate each tube with 0 1 mL of the overnight culture Grow the cultures at 37 C with vigorous shaking to an ODg o9 0 5 the cells should be in mid log phase While the cells are growing prepare four 10 fold serial dilutions of 20 arabinose with sterile water using aseptic technique e g 2 0 2 0 02 and 0 002 Remove a 1 mL aliquot of cells from each tube centrifuge at maximum speed in a microcentrifuge for 30 seconds and aspirate the supernatant Freeze the cell pellet at 20 C This is the zero time point sa
6. 1980 The Escherichia coli L Arabinose Operon Binding Sites of the Regulatory Proteins and a Mechanism of Positive and Negative Regulation Proc Natl Acad Sci USA 77 3346 3350 Russell C B Stewart R C and Dahlquist F W 1989 Control of Transducer Methylation Levels in Escherichia coli Investigation of Components Essential for Modulation of Methylation and Demethylation Reactions J Bacteriol 171 3609 3618 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press San Millan J L Boyd D Dalbey R Wickner W and Beckwith J 1989 Use of phoA Fusions to Study the Topology of the Escherichia coli Inner Membrane Protein Leader Peptidase J Bacteriol 171 5536 5541 Schleif R S 1992 DNA Looping Ann Rev Biochem 61 199 223 Shuman S 1994 Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase J Biol Chem 269 32678 32684 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Wallace B J and Kushner S R 1984 Genetic and Physical Analysis of the Thioredoxin trxA Gene of Escherichia coli K 12 Gene 32 399 408
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8. The vector PCR insert reaction should yield hundreds of colonies To analyze the transformations isolate plasmid DNA and digest with Hind III or another appropriate restriction enzyme The table below lists the Hind III digestion pattern that you should see for inserts that are cloned in the correct orientation or in the reverse orientation Vector Restriction Enzyme Expected Digestion Pattern bp pBAD202 D TOPO Hind III Correct orientation 560 4642 Reverse orientation 226 4976 Empty vector 4448 pUC19 plasmid is included as a control to check the transformation efficiency of One Shot TOP10 competent cells Transform one vial of One Shot TOP10 cells with 10 pg of pUC19 using the protocol on page 13 or 14 Plate 10 uL of the transformation mixture plus 20 uL of S O C on LB plates containing 100 pg mL ampicillin Transformation efficiency should be 1 x 10 cfu ug DNA 29 Map and Features of pBAD202 D TOPO Map of The map below shows the features of pBAD202 D TOPO The complete pBAD202 sequence of the vector is available for downloading at www invitrogen com or D TOPO from Technical Support page 36 Neo Sac Hind Il HP thio 7 ccc TT AAG GGC p 2 redoxin EK Site Meath pes V5 epitope 6x His Stop pBAD202 D TOPO 4448 nucleotides Arabinose O2 operator region 4 19 Arabinose O4 operator region 161 182 CAP binding site 203 216 Arabinose l4 and l2 reg
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10. AraC dimer contacts the O2 and I half sites of the araBAD operon forming a 210 bp DNA loop see the figure below For maximum transcriptional activation two events are required e Arabinose binds to AraC and causes the protein to release the O site and bind the D site which is adjacent to the I site This releases the DNA loop and allows transcription to begin e The cAMP activator protein CAP cAMP complex binds to the DNA and stimulates binding of AraC to I and h AraC dimer No transcription arabinose Transcription Glucose Basal expression levels can be repressed by introducing glucose to the growth Repression medium Glucose acts by lowering cAMP levels which in turn decreases the binding of CAP As cAMP levels are lowered transcriptional activation is decreased 33 Accessory Products Additional Products Proofreading DNA Polymerases Electrocompetent Cells 34 Many of the reagents supplied with the pBAD Directional TOPO Expression Kit and other reagents suitable for use with the kit are available separately from Invitrogen Ordering information for these reagents is provided below For details visit www invitrogen com Product Amount Cat no PCR Optimizer Kit 100 reactions K1220 01 PCR SuperMix High Fidelity 100 reactions 10790 020 One Shot TOP10 Chemically Competent 10 reactions C4040 10 cole 20 reactions 4040 03 P
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12. contains a pUC origin they act as low copy number plasmids resulting in lower yields of the vectors How Directional TOPO Cloning Works How Topoisomerase Works Directional TOPO Cloning Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5 CCCTT in one strand Shuman 1991 The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3 phosphate of the cleaved strand and a tyrosyl residue Tyr 274 of topoisomerase I The phospho tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5 hydroxyl of the original cleaved strand reversing the reaction and releasing topoisomerase Shuman 1994 TOPO Cloning exploits this reaction to efficiently clone PCR products Directional joining of double strand DNA using TOPO charged oligonucleotides occurs by adding a 3 single stranded end overhang to the incoming DNA Cheng and Shuman 2000 This single stranded overhang is identical to the 5 end of the TOPO charged DNA fragment At Invitrogen this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO charged DNA and adapting it to a whole vector format In this system PCR products are directionally cloned by adding four bases to the forward primer CACC The overhang in the cloning vector GTGG invades the 5 end of the PCR product anneals
13. information on the mechanism of expression and repression of the ara regulon see page 33 or refer to Schleif 1992 The 11 7 kDa thioredoxin protein is found in yeast plants and mammals as well as in bacteria It was originally isolated from E coli as a hydrogen donor for ribonuclease reductase see Holmgren 1985 for a review The gene has been completely sequenced Wallace and Kushner 1984 The protein has been crystallized and its three dimensional structure determined Katti et al 1990 When overexpressed in E coli thioredoxin is able to accumulate to approximately 40 of the total cellular protein and still remains soluble When used as a fusion partner thioredoxin can increase translation efficiency and in some cases solubility of eukaryotic proteins expressed in E coli Examples of eukaryotic proteins that have been produced as soluble C terminal fusions to the thioredoxin protein in E coli LaVallie et al 1993 include e Murine interleukin 2 e Human interleukin 3 e Murine interleukin 4 e Murine interleukin 5 e Human macrophage colony stimulating factor e Murine steel factor e Murine leukemia inhibitory factor e Human bone morphogenetic protein 2 The thioredoxin protein has been mutated to contain a metal binding domain and is termed His Patch thioredoxin To create a metal binding domain in the thioredoxin protein the glutamate residue at position 32 and the glutamine residue at position 64
14. pages 26 27 e Estimate the concentration of your PCR product Use this information when setting up your TOPO Cloning reaction see Amount of PCR Product to Use in the TOPO Cloning Reaction next page for details Performing the TOPO Cloning Reaction Introduction Amount of PCR Product to Use in the TOPO Cloning Reaction Once you have produced the desired PCR product you are ready to TOPO Clone it into the pBAD202 D TOPO vector and transform the recombinant vector into One Shot TOP10 E coli It is important to have everything you need set up and ready to use to ensure best results We suggest that you read this section and the section entitled Transforming One Shot TOP10 Competent Cells pages 12 14 before beginning If this is the first time you have TOPO Cloned perform the control reactions on pages 28 29 in parallel with your samples When performing directional TOPO Cloning we have found that the molar ratio of PCR product TOPO vector used in the reaction is critical to its success To obtain the highest TOPO Cloning efficiency use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector see figure below Note that the TOPO Cloning efficiency decreases significantly if the ratio of PCR product TOPO vector is lt 0 1 1 or gt 5 1 These results are generally obtained if too little PCR product is used i e PCR product is too dilute or if too much PCR product is used in the TOPO Cloning reac
15. reaction as directed below Use the procedure below to perform the TOPO Cloning reaction Set up the TOPO Cloning reaction depending on whether you plan to transform chemically competent E coli or electrocompetent E coli For optimal results use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector in your TOPO Cloning reaction Note The blue color of the TOPO vector solution is normal and is used to visualize the solution Reagents Chemically Competent Electrocompetent E coli E coli Fresh PCR product 0 5 to4 uL 0 5 to4 uL Salt Solution 1 pL Dilute Salt Solution 1 4 1 uL Sterile Water add to a final volume of 5 uL add to a final volume of 5 uL TOPO vector 1 pL 1 pL Final volume 6 pL 6 uL Store all reagents at 20 C when finished Store salt solutions and water at room temperature or 4 C 1 Mix reaction gently and incubate for 5 minutes at room temperature 22 23 C Note For most applications 5 minutes yields plenty of colonies for analysis Depending on your needs you can vary the length of the TOPO Cloning reaction from 30 seconds to 30 minutes For routine subcloning of PCR products 30 seconds may be sufficient For large PCR products gt 1 kb or if you are TOPO Cloning a pool of PCR products increasing the reaction time may yield more colonies 2 Place the reaction on ice and proceed to Transforming One Shot TOP10 Competent Cells next page Note
16. to the added bases and stabilizes the PCR product in the correct orientation Inserts can be cloned in the correct orientation with efficiencies equal to or greater than 90 Topoisomerase oe CCCrL CACC ATG NNN NNN AAG GG GGGAAGTGG GTGG TAC NNN NNN ITO CC PCR product Overhang Overhang invades double stranded 0 DNA displacing the bottom strand Topoisomerase CCCTTCACC ATG NNN NNN AAG GG GGGAAGTGG TAC NNN NNN TTC CC Arabinose Regulation and Thioredoxin Regulation of Expression by Arabinose Thioredoxin His Patch Thioredoxin In the presence of arabinose expression from Paap is induced while only very low levels of transcription are observed from Pran in the absence of arabinose Lee 1980 Lee et al 1987 Uninduced levels are repressed even further by growth in the presence of glucose 0 176 to 0 276 Glucose reduces the levels of 3 5 cyclic AMP lowering expression from the catabolite repressed Paap promoter Miyada et al 1984 By varying the concentration of arabinose protein expression levels can be optimized to ensure maximum expression of protein In addition the tight regulation of Prap by AraC is useful for expression of potentially toxic or essential genes Carson et al 1991 Dalbey and Wickner 1985 Guzman et al 1992 Kuhn and Wickner 1985 Russell et al 1989 San Millan et al 1989 For more
17. were mutated to histidine residues When His Patch thioredoxin folds the histidines at positions 32 and 64 interact with a native histidine at position 8 to form a patch This histidine patch has been shown to have high affinity for divalent cations Lu et al 1996 His Patch thioredoxin HP thioredoxin proteins can therefore be purified on metal chelating resins e g ProBond Experiment Outline Experiment Outline The table below describes the general steps needed to clone and express your gene of interest For more details refer to the pages indicated Step Action Page 1 Design PCR primers to clone your gene of interest in frame with the N terminal His Patch thioredoxin and C terminal V5 epitope and polyhistidine tag if desired Consult the diagram on page 8 to help you design your PCR primers 5 8 Produce your blunt end PCR product TOPO Clone your PCR product into pBAD202 D TOPO and transform into One Shot TOP10 E coli Select transformants on LB plates containing the appropriate antibiotic 10 14 Analyze transformants by restriction digestion or PCR 15 Select a transformant with the correct restriction pattern and sequence it to confirm that your gene is cloned in frame with the N terminal His Patch thioredoxin and C terminal V5 epitope and polyhistidine tag if desired 15 Select positive transformant and induce expression with arabinose 17 18
18. 15907 15913 LaVallie E R DiBlasio E A Kovacic S Grant K L Schendel P F and McCoy J M 1993 A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E coli Cytoplasm Bio Technology 11 187 193 Lee N 1980 Molecular Aspects of ara Regulation In The Operon J H Miller and W S Reznikoff eds Cold Spring Harbor N Y Cold Spring Harbor Laboratory pp 389 410 Lee N Francklyn C and Hamilton E P 1987 Arabinose Induced Binding of AraC Protein to aral Activates the araBAD Operon Promoter Proc Natl Acad Sci USA 84 8814 8818 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Continued on next page 39 References Continued Lu Z DiBlasio Smith E A Grant K L Warne N W LaVallie E R Collins Racie L A Follettie M T Williamson M J and McCoy J M 1996 Histidine Patch Thioredoxins J Biol Chem 271 5059 5065 Miyada C G Stoltzfus L and Wilcox G 1984 Regulation of the araC Gene of Escherichia coli Catabolite Repression Autoregulation and Effect on araBAD Expression Proc Natl Acad Sci USA 81 4120 4124 Ogden S Haggerty D Stoner C M Kolodrubetz D and Schleif R
19. 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany Continued on next page 37 Purchaser Notification Continued Limited Use Label License No 29 ThioFusion Information for European Customers 38 The ThioFusion Expression System is licensed under U S Patent No 5 270 181 from Genetics Institute Inc for research use only Licenses for commercial manufacture or use may be obtained directly from Genetics Institute Inc 87 Cambridgepark Drive Cambridge MA 02140 The LMG194 cell line is genetically modified As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Brownstein M J Carpten J D and Smith J R 1996 Modulation of Non Templated Nucleotide Addition by Tag DNA Polymerase Primer Modifications that Facilitate Genotyping BioTechniques 20 1004 1010 Carson M J Barondess J J and Beckwit
20. AD202 D TOPO vector 15 20 ng uL plasmid DNA in 20 pL 50 glycerol 50 mM Tris HCl pH 7 4 at 25 C 1mM EDTA 1 mM DTT 0 1 Triton X 100 100 pg mL BSA 30 M bromophenol blue dNTP Mix 12 5 mM dATP 12 5 mM dCTP 10 pL 12 5 mM dGTP 12 5 mM dTTP in water pH 8 Salt Solution 1 2 M NaCl 50 uL 0 06 M MgCl Sterile Water 1 mL 20 L Arabinose 20 in sterile water 1 mL TrxFus Forward Sequencing Primer 0 1 ug uL in TE Buffer pH 8 20 pL pBAD Reverse Sequencing Primer 0 1 pg L in TE Buffer pH 8 20 uL Control PCR Primers 0 1 ug uL each in TE Buffer pH 8 10 pL Control PCR Template 0 1 ug uL in TE Buffer pH 8 10 pL pBAD202 D lacZ Expression 0 01 pg pL in TE buffer pH 8 10 uL Control Plasmid The table below provides the sequences of the Trx Forward and pBAD Reverse sequencing primers Two micrograms of each primer are supplied Primer Sequence pMoles Supplied TrxFus Forward 5 TITCCITCGACGCTAACCTG 3 371 pBAD Reverse 5 GATITAATCIGTATCAGG 3 363 Continued on next page Kit Contents and Storage Continued One Shot TOP10 Reagents Genotype of TOP10 Genotype of LMG194 Preparing LMG194 Glycerol Stocks vi The table below lists the items included in the One Shot TOP10 Chemically Competent E coli kit Box 2 Transformation efficiency is 1 x 10 cfu ug DNA Store Box 2 at 80 C Item Composition Amount TOP10 cells 21 x 50 uL S O C Medium 2
21. Autoclave on liquid cycle for 20 minutes Allow solution to cool to 55 C and add antibiotic if needed 4 Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes 3 After autoclaving cool to 55 C add antibiotic if needed and pour into 10 cm plates 4 Let the plates harden then invert them and store at 4 C in the dark 2 Tryptone 0 5 Yeast Extract 0 05 NaCl 2 5 mM KCl 10 mM MgCl 1 Dissolve 20 g tryptone 5 g yeast extract and 0 5 g NaCl in 950 mL deionized water 2 Make a 250 mM KCI solution by dissolving 1 86 g of KCl in 100 mL of deionized water Add 10 mL of this stock KCI solution to the solution in Step 1 3 Adjust pH to 7 5 with 5 M NaOH and add deionized water to 1 liter Autoclave this solution cool to 55 C and add 10 mL of sterile 1 M MgCL You may also add antibiotic if needed 5 Store the medium at 4 C Medium is stable for only 1 2 weeks Continued on next page Recipes Continued RM Medium Glucose 10X M9 Salts Lysis Buffer 1X M9 Salts see below for recipe for 10X M9 Salts 2 Casamino Acids 0 2 glucose antibiotic to the appropriate concentration 1 For 1 liter of RM medium mix 20 g Casamino Acids and 890 mL deionized water Autoclave 20 minutes on liquid cycle After the autoclaved solution has cooled add the following sterile solutions
22. Example 2 on the next page e If you do NOT wish to fuse your PCR product in frame with the C terminal V5 epitope tag and polyhistidine region include the native sequence containing the stop codon in the reverse primer or make sure the stop codon is upstream from the reverse PCR primer binding site see Example 2 on the next page Below is the sequence of the C terminus of a theoretical protein You want to fuse the protein in frame with a C terminal tag The stop codon is underlined DNA sequence AAG TCG GAG CAC TCG ACG ACG GTG TAG 3 One solution is to design the reverse PCR primer to start with the codon just up stream of the stop codon but the last two codons contain GTGG underlined below which is identical to the 4 bp overhang sequence As a result the reverse primer will be complementary to the 4 bp overhang sequence increasing the probability that the PCR product will clone in the opposite orientation You want to avoid this situation DNA sequence AAG TCG GAG CAC TCG ACG ACG GTG TAG 3 Proposed Reverse PCR primer sequence TG AGC TGC TGC CAC AAA 5 Another solution is to design the reverse primer so that it hybridizes just down stream of the stop codon but still includes the C terminus of the ORF Note that you need to replace the stop codon with a codon for an innocuous amino acid such as glycine alanine or lysine Continued on next page Designing PCR Primers Continued Example 2 of Reverse Primer De
23. Invitrogen pBAD Directional TOPO Expression Kit Five minute directional TOPO cloning of blunt end PCR products into vectors for soluble regulated expression and purification in E coli Catalog no K4202 01 Revision date 7 June 2010 Manual part no 25 0526 MAN0000286 Contents Kit Contents and Storage ose e aee ede ep etes dte di eire d I Dee etes ie pi eei ertet v Introduction earned reen aenema aren 1 Description of the System diete biete iden akten nen kedeng aa nee ebenso sese eter RE Doe e obvio esie sechsten 1 How Directional TORO Cloning Works tnt etn 2 Arabinose Regulation and Thioredoxin sse tette tenente nennen 3 Experiment Ot tlirie 6e e RH DH IHRER Gne RAT RUN renbaan eti etg ree Lee hte ded 4 LugQg rr 5 D signing PCR Primers iesire seamen on eid aee tte istae Pee e FE ee pres fert oie eae e AEE EAS EESE 5 Producing Blunt End PCR Products nnee senen anenenenenenenenenensenenenrnenennenenenenevenenenenenenenenenensenenenenen 9 Performing the TOPO Cloning Reaction iis aen inen eni temet ben Sd bui etait tie 10 Transforming One Shot TOPIO Competent Cells aieo desees d itte venae 12 Analyzing Transformants nnnnsnsenenensaneneneneneneneneneneneneneneneneenenenenenensnenenensenenenenenenennenenenenenenenenenensenenenenene 15 Expressing the PCR Product 0 3 45e eet an oh aen oons ante al Mie Banse bean eea ese es aded 17 Analyzing Samples nina sey antari handed a qued edid eed
24. NA Polymerase AccuPrime Pfx DNA Polymerase or Pfx50 DNA Polymerase available separately from Invitrogen see page 34 for ordering information Follow the guidelines below to produce your blunt end PCR product e Thermocycler and thermostable proofreading polymerase e 10X PCR buffer appropriate for your polymerase DNA template and primers for PCR product Note dNTPs adjusted to pH 8 are provided in the kit Set up a 25 uL or 50 uL PCR reaction using the guidelines below e Follow the instructions and recommendations provided by the manufacturer of your thermostable proofreading polymerase to produce blunt end PCR products e Use the cycling parameters suitable for your primers and template Make sure to optimize PCR conditions to produce a single discrete PCR product e Usea 7 to 30 minute final extension to ensure that all PCR products are completely extended e After cycling place the tube on ice or store at 20 C for up to 2 weeks Proceed to Checking the PCR Product below After you have produced your blunt end PCR product use agarose gel electrophoresis to verify the quality and quantity of your PCR product Check for the following outcomes below e Be sure you have a single discrete band of the correct size If you do not have a single discrete band follow the manufacturer s recommendations to optimize your PCR with the polymerase of your choice Alternatively gel purify the desired product see
25. Proteins Note 20 To determine the success of your expression experiment perform the following types of analyses 1 Stain the polyacrylamide gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein Use the uninduced culture as a negative control 2 Perform a western blot to confirm that the overexpressed band is your desired protein see below this is the preferred method for analyzing your samples 3 Use the expression control plasmid to confirm that growth and induction were performed properly The size of the B galactosidase fusion protein expressed from the positive control plasmid when induced with 0 02 arabinose is approximately 133 kDa 4 Determine the approximate arabinose concentration for maximum expression To detect expression of your recombinant fusion protein by western blot analysis you may use antibodies against the appropriate epitope see page 35 for ordering information or an antibody to your protein of interest In addition the Positope Control Protein is available from Invitrogen for use as a positive control for detection of fusion proteins containing a thioredoxin V5 or C terminal 6xHis epitope The ready to use WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods For more information refer to www
26. Tryptone 6 mL may be stored at room 0 5 Yeast Extract temperature or 4 C 10 mM NaCl 2 5 mM KCl 10 mM MgCl 10 mM MgSO 20 mM glucose pUC19 Control DNA 10 pg uL in 50 uL 5 mM Tris HCl 0 5 mM EDTA pH 8 0 Use this strain for general cloning of blunt end PCR products into the pBAD202 D TOPO vector Genotype F mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG F AlacX74 galE thi rpsL AphoA Pou II Aara714 leu Tn10 Note This strain is deleted for araBADC It is also streptomycin and tetracycline resistant Store the LMG194 E coli stab supplied with the kit at 4 C Upon receipt we recommend that you prepare a set of LMG194 glycerol master stocks within two weeks of receiving the kit 1 Streak a small portion of the LMG194 cells from the stab on an LB plate containing the appropriate antibiotics and incubate at 37 C overnight 2 Isolate a single colony and inoculate into 5 10 mL of LB medium with the appropriate antibiotics 3 Grow the culture to stationary phase OD 1 2 4 Mix 0 8 mL of culture with 0 2 mL of sterile glycerol and transfer to a cryovial Store at 80 C Use one master stock to create working stocks for regular use Introduction Description of the System Product Features pBAD202 D TOPO Vector The pBAD Directional TOPO Expression Kit utilizes a highly efficient 5 minute cloning strategy TOPO
27. Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu TrxFus Forward priming site NgoM Nae EK recognition site EK cleavage site 655 TTC CTC GAC GCT AAC CTG GCC GGC TCT GGA TCC GGT GAT GAC GAT GAC AAG CTG GGA ATT GAT CCC Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Gly Ile Asp Pro Sac Hind Ill V5 epitope 721 TT AAG GGC GAG crc AAG CTT GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC AAG TGG TTC CCG Phe Thy Lys Gly Glu Leu Lys Leu Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Polyhistidine 6xHis region Pme GAT TCT acc m mom 781 GAT TCT ACG CGT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACGG TCTCCAGCTT GGCTGTTTTG Asp Ser Thr Arg Thr Gly His His His His His His pBAD Reverse priming site 850 GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC rrnB T1 and T2 transcriptional terminator 930 GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC 1010 TCCCCATGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC Producing Blunt End PCR Products Introduction Materials Supplied by the User Producing Blunt End PCR Products Checking the PCR Product After you have decided on a PCR strategy and have synthesized the primers produce your blunt end PCR product using any thermostable proofreading polymerase We recommend Platinum Pfx D
28. You may store the TOPO Cloning reaction at 20 C overnight 11 Transforming One Shot TOP10 Competent Cells Introduction Materials Supplied by the User Preparing for Transformation Once you have performed the TOPO Cloning reaction you will transform your pBAD202 D TOPC construct into competent E coli One Shot TOP10 Chemically Competent E coli are included with the kit to facilitate transformation however you may also transform electrocompetent cells This section provides protocols to transform chemically competent or electrocompetent E coli e 42 C water bath or electroporator with cuvettes optional e LB plates containing 50 pg mL kanamycin two for each transformation e 37 C shaking and non shaking incubator For each transformation you will need one vial of competent cells and two selective plates e Equilibrate a water bath to 42 C for chemical transformation or set up your electroporator if you are using electrocompetent E coli e Warm the vial of S O C medium from Box 2 to room temperature Warm LB plates containing 50 ug mL kanamycin at 37 C for 30 minutes e Thaw on ice 1 vial of One Shot TOP10 cells from Box 2 for each transformation Continued on next page Transforming One Shot TOP10 Competent Cells Continued Important One Shot TOP10 Chemical Transformation Protocol The number of colonies obtained after transforming the pBAD202 D TOPO vector int
29. ains artifacts i e does not run as a single discrete band on an agarose gel e Optimize your PCR using the proofreading polymerase of choice e _Gel purify your PCR product to remove primer dimers and smaller PCR products Incorrect PCR primer design e Make sure that the forward PCR primer contains the sequence CACC at the 5 end e Make sure that the reverse PCR primer does not contain the sequence CACC at the 5 end Few or no colonies obtained from sample reaction and the transformation control gave no colonies One Shot competent E coli stored incorrectly Store One Shot competent E coli at 80 C If you are using another E coli strain follow the manufacturer s instructions One Shot transformation protocol not followed correctly Follow the One Shot transformation protocol provided on page 13 or page 14 Insufficient amount of E coli plated Increase the amount of E coli plated Transformants plated on selective plates containing the wrong antibiotic Use the appropriate antibiotic for selection 23 Recipes LB Luria Bertani Medium and Plates SOB Medium 24 Appendix Composition 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 mL deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter 3
30. aseptically 10X M9 Salts 100 mL 1M MgCl 1 mL 20 glucose 10 mL antibiotic 4 Mix well and store medium containing antibiotic at 4 C Medium is good for 1 month at 4 C For 1 liter Na HPO 60 8 KHPO 30 8 NaCl 5g NH Cl 10g Water 900 mL 1 Dissolve reagents in the water and adjust the pH to 7 4 with 10 M NaOH 2 Add water to 1 liter and autoclave for 20 minutes on liquid cycle 3 Store the solution at room temperature 50 mM potassium phosphate pH 7 8 400 mM NaCl 100 mM KCI 1096 glycerol 0 5 Triton X 100 10 mM imidazole 1 Prepare 1 M stock solutions of KH PO and K3HPO 2 For100 mL dissolve the following reagents in 90 mL of deionized water 4 7 mL KHPO 2 3 g NaCI 0 75 g KCI 10 mL glycerol 0 5 mL Triton X 100 68 mg imidazole 3 Mix thoroughly and adjust pH to 7 8 with HCI Bring the volume to 100 mL 4 Store the buffer at 4 C 25 Purifying the PCR Products Introduction Using the PureLink Quick Gel Extraction Kit 26 Smearing multiple banding primer dimer artifacts or large PCR products gt 3 kb may necessitate gel purification If you intend to purify your PCR product be extremely careful to remove all sources of nuclease contamination There are many protocols to isolate DNA fragments or remove oligonucleotides Refer to Current Protocols in Molecular Biology Unit 2 6 Ausubel et al 1994 for the most common protocols Two simple protocols are described below
31. ast 1 hour at 37 C to allow expression of the antibiotic resistance gene Spread 50 100 uL from each transformation on a prewarmed selective plate and incubate overnight at 37 C We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies An efficient TOPO Cloning reaction may produce several hundred colonies Pick 5 colonies for analysis see Analyzing Positive Clones page 15 To prevent arcing of your samples during electroporation the volume of cells should be between 50 uL and 80 uL 0 1 cm cuvettes or 100 uL to 200 uL 0 2 cm cuvettes If you experience arcing during transformation try one of the following suggestions Reduce the voltage normally used to charge your electroporator by 1076 Reduce the pulse length by reducing the load resistance to 100 ohms Ethanol precipitate the TOPO Cloning reaction and resuspend in water prior to electroporation Analyzing Transformants Analyzing Positive 1 Clones Sequencing Important 2 Pick 5 colonies and culture them overnight in LB or SOB medium containing 50 ug mL kanamycin Isolate plasmid DNA using your method of choice We recommend using the PureLink HO Mini Plasmid Purification or PureLink HiPure Plasmid Miniprep kits see page 34 for ordering information Refer to www invitrogen com or contact Technical Support for more information on a large selection of plasmid purification columns N
32. cin 2 Isolate a single colony and inoculate into 1 2 mL of LB containing 50 pL mL kanamycin 3 Grow until culture reaches stationary phase Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C Expressing the PCR Product Introduction Using LMG194 Plasmid Preparation Positive Control Basic Strategy Because each recombinant protein has different characteristics that may affect optimal expression it is helpful to vary the arabinose concentration and or run a time course of expression to determine the best conditions for optimal expression of your particular protein The E coli strain LMG194 Guzman et al 1995 is included in the kit to allow additional repression for low basal level expression of toxic genes This strain is capable of growth on minimal medium RM medium which allows repression of Psap by glucose After you have determined that you have the correct construct transform it into LMG194 prior to performing expression experiments Follow the guidelines below for using LMG194 e Induce the pBAD promoter when cells are growing in LB or RM Glucose e Ifyou are growing your construct under maximal repression i e with D glucose in RM media then you must spin down the culture and resuspend it in RM containing 0 2 glycerol and Arabinose i e substitute glycerol for the glucose in the media recipe on page 25 You may prepare plasmid DNA usin
33. d directionally TOPO Cloned into pBAD202 D TOPO in frame with HP thioredoxin and the C terminal peptide containing the V5 epitope and a polyhistidine 6xHis tag The size of the B galactosidase fusion protein is approximately 133 kDa Map of The figure below shows the elements of pBAD202 D lacZ The complete pBAD202 D lacZ sequence of the vector is available at www invitrogen com or by contacting Technical Support page 36 2 FEET lacz W5 epitope 6x His fStop E pBAD202 D lacZ 7 520 nucleotides Arabinose O operator region 4 19 Arabinose O4 operator region 161 182 CAP binding site 203 216 Arabinose l4 and l2 region 213 251 Arabinose minimal promoter 248 276 Ribosome binding site 329 332 His Patch Thioredoxin ORF 346 674 TrxFus Forward priming site 655 672 Enterokinase recognition site 691 705 lacZ ORF 730 3801 V5 epitope 3820 3861 Polyhistidine 6xHis region 3871 3888 pBAD Reverse priming site 3944 3961 rrnB transcriptional termination region 3994 4151 Kanamycin resistance gene c 4520 5314 pUC origin 5412 6085 araC ORF c 6616 7494 c complementary strand 32 Regulation by Arabinose Regulation of the The araBAD promoter used in the pBAD D TOPO vectors is both positively Paap Promoter and negatively regulated by the product of the araC gene Ogden et al 1980 Schleif 1992 AraC is a transcriptional regulator that forms a complex with L arabinose In the absence of arabinose the
34. e sample in dry ice or liquid nitrogen and then thaw it at 42 C Repeat 2 to 3 times Note To facilitate lysis you may add lysozyme to the sample or sonicate the cells 3 Centrifuge samples at maximum speed in a microcentrifuge for 1 minute at 4 C to pellet insoluble proteins Transfer supernatant to a fresh tube and store on ice 4 Mixtogether equivalent amounts of supernatant and 2X SDS PAGE sample buffer and boil for 5 minutes 5 Add 500 uL of 1X SDS PAGE sample buffer to the pellets from Step 3 and boil 5 minutes 6 Load 10 uL of the supernatant sample and 5 uL of the pellet sample onto an SDS PAGE gel and electrophorese To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels and electrophoresis apparatus are available from Invitrogen The NuPAGE Gel System avoids the protein modifications associated with LaemmLi type SDS PAGE ensuring optimal separation for protein analysis In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits For more information about the appropriate gels standards and stains to use to visualize your recombinant protein refer to www invitrogen com or contact Technical Support page 36 Continued on next page 19 Analyzing Samples Continued Analyzing Samples Detecting Recombinant Fusion
35. em ae iier oe latc dad edes 19 Troubleshooting ene e re etit eer de pied etes a rie cannon ped d p inis repr ei eres 22 AppendiX OT 24 RECIPES unies eoa boim e poe E adis getan aii indt etii teti Uri adest eir ta 24 Purifying the PCR Prod cts un E ite 26 Performing the Control Reactions insistir e ise AEN E ie iE n TEE E iR E E Ee Eie iE 28 Map and Features of ppBAD202 D TOPO nanasan eeen iet 30 Map of PBAD202 D MEZ irte or ceo i trie e eee on id rne dn 32 Regulation by Arabinose ient ite e IR HEEL ee EHE Iren He RS PEE E SH pedea 33 Accessory Products snos Cane edet fee iie ed ilte dt e e Gosapadveh sedeastevdashenshens 34 Technical Support eniin deeem e deitub eique etnia ut and d 36 PurchaserNotficationcusedec ema emtten E ea AA T E a A tte 37 References eee ean eaae dehet a te cen ee a a a e eeen 39 Kit Contents and Storage Shipping Storage pBAD D TOPO Reagents Sequences of the Primers The pBAD Directional TOPO Expression Kit is shipped on dry ice Each kit contains a box with pBAD D TOPO reagents Box 1 a box with One Shot TOP10 Chemically Competent E coli Box 2 and a stab of LMG194 Store Box 1 at 20 C and Box 2 at 80 C Store the LMG194 stab at 4 C pBAD D TOPO reagents Box 1 are listed below Note that you must supply a thermostable proofreading polymerase and the appropriate PCR buffer Store Box 1 at 20 C Item Concentration Amount pB
36. en vector encoded amino acids remaining at the N terminus of the protein see page 8 A recombinant preparation of the catalytic subunit of bovine enterokinase EKMax is available from Invitrogen Instructions for digestion are included with the product To remove EKMax from the digest you may use EK Away Resin also available from Invitrogen see page 34 for ordering information 21 Troubleshooting TOPO Cloning Reaction and Transformation The table below lists some potential problems and possible solutions that may help you troubleshoot the TOPO Cloning and transformation reactions To help evaluate your results we recommend that you perform the control reactions in parallel with your samples see pages 28 29 Problem Reason Solution Few or no colonies Suboptimal ratio of PCR Use a 0 5 1 to 2 1 molar ratio of PCR obtained from sample product TOPO vector used in the product TOPO vector reaction and the TOPO Cloning reaction ra alc control Too much PCR product used in the e Dilute the PCR product ave colonies E TOPO Cloning reaction e Usea0 5 1 to 2 1 molar ratio of PCR product TOPO vector PCR product too dilute e Concentrate the PCR product Use a 0 5 1 to 2 1 molar ratio of PCR product TOPO vector PCR primers contain 5 phosphates Do not add 5 phosphates to your PCR primers Incorrect PCR primer design Make sure that the forward PCR primer contains the
37. ension 1 minute 72 C Final Extension 7 minutes 72 C 1X 3 Remove 10 uL from the reaction and analyze by agarose gel electrophoresis A discrete 750 bp band should be visible 4 Estimate the concentration of the PCR product and adjust the concentration as necessary such that the amount of PCR product used in the control TOPO Cloning reaction results in an optimal molar ratio of PCR product TOPO vector i e 0 5 1 to 2 1 Proceed to the Control TOPO Cloning Reactions next page Continued on next page 28 Performing the Control Reactions Continued Control TOPO Cloning Reactions Analyzing Results Transformation Control Using the control PCR product produced on the previous page and the pBAD D TOPO vector set up two 6 uL TOPO Cloning reactions as described below 1 Set up control TOPO Cloning reactions Reagent Vector Only Vector PCR Insert Sterile Water 4 ul 3 pL Salt Solution 1 uL 1 pL Control PCR Product 1 uL pBAD D TOPO vector 1 pL 1 pL Final volume 6 pL 6 pL Incubate at room temperature for 5 minutes and place on ice Transform 3 uL of each reaction into separate vials of One Shot TOP10 competent cells page 13 or 14 4 Spread 100 200 uL of each transformation mix onto LB plates containing 50 pg mL kanamycin Plate two different volumes to ensure that at least one plate has well spaced colonies 5 Incubate overnight at 37 C
38. etric detection methods The amount of antibody supplied is sufficient for 25 Western blots Antibody HHHHHH COOH Product Epitope Cat no Anti Thio Antibody Detects His Patch thioredoxin R920 25 fusion proteins Note The exact epitope detected by this antibody has not been mapped Anti V5 Antibody Detects 14 amino acid epitope R960 25 Anti V5 HRP Antibody derived from the P and V proteins R961 25 gt of the paramyxovirus SV5 Anti V5 AP Antibody Southern et al 1991 R962 25 GKPIPNPLLGLDST Anti His C term Antibody Detects the C terminal R930 25 Anti His C term HRP polyhistidine 6xHis tag requires R931 25 Antibody the free carboxyl group for TUE detection Lindner et al 1997 Anti His C term AP R932 25 If your gene of interest in is frame with the C terminal peptide containing the polyhistidine 6xHis tag you may use Invitrogen s ProBond or Ni NTA Purification System to purify your recombinant fusion protein See the table below for ordering information 10 mL polypropylene columns Product Amount Cat no ProBond Purification System 6 purifications K850 01 ProBond Nickel Chelating Resin 50mL R801 01 150 mL R801 15 Ni NTA Purification System 6 purifications K950 01 Ni NTA Agarose 10 mL R901 01 25 mL R901 15 Purification Columns 50 columns R640 50 35 Technical Support Web Resources Visit the
39. f you wish to include the N terminal thioredoxin design the forward PCR primer to ensure that your protein is in frame with the N terminal leader peptide Below is the DNA sequence of the N terminus of a theoretical protein and the proposed sequence for your forward PCR primer DNA sequence 5 GTA GGA TCT GAT AAA Proposed Forward PCR primer 5 C ACC GTA GGA TCT GAT AAA The first three base pairs of the PCR product following the 5 CACC overhang will constitute a functional codon Continued on next page Designing PCR Primers Continued Guidelines to Design the Reverse Primer Example 1 of Reverse Primer Design When designing your reverse PCR primer consider the points below Refer to page 8 for a diagram of the TOPO Cloning site on the pBAD202 D TOPO vector e To ensure that your PCR product clones directionally with high efficiency the reverse PCR primer MUST NOT be complementary to the overhang sequence GTGG at the 5 end A one base pair mismatch can reduce the directional cloning efficiency from 90 to 50 increasing the likelihood of your ORF cloning in the opposite orientation see Example 1 below We have not observed evidence of PCR products cloning in the opposite orientation from a two base pair mismatch e To fuse your PCR product in frame with the C terminal V5 epitope tag and polyhistidine region design the reverse PCR primer to remove the native stop codon in the gene of interest see
40. for downloading at www invitrogen com or by contacting Technical Support page 36 O2 Region 1 AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT TCTCGCTAAC CAAACCGGTA 81 ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA O1 Region CAP binding site 161 CGGCAGAAAA GTCCACATTG ATTATTTGCA CGGCGTCACA CTTTGCTATG CCATAGCATT TTTATCCATA AGATTAGCGG 35 10 12 and 11 Region 241 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTTGGG CTAGAAATAA TTTTGTTTAA RBS Neo His patch HP thioredoxin site 321 CTTTAAGAAG GAGATATACA TACCC ATG GGA TCT GAT AAA ATT ATT CAT CTG ACT GAT GAT TCT TTT GAT Met Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp 391 ACT GAT GTA CTT AAG GCA GAT GGT GCA ATC CTG GTT GAT TTC TGG GCA CAC TGG TGC GGT CCG TGC Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala His Trp Cys Gly Pro Cys 457 AAA ATG ATC GCT CCG ATT CTG GAT GAA ATC GCT GAC GAA TAT CAG GGC AAA CTG ACC GTT GCA AAA Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys 523 CTG AAC ATC GAT CAC AAC CCG GGC ACT GCG CCG AAA TAT GGC ATC CGT GGT ATC CCG ACT CTG CTG Leu Asn Ile Asp His Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 589 CTG TTC AAA AAC GGT GAA GTG GCG GCA ACC AAA GTG GGT GCA CTG TCT AAA GGT CAG TTG AAA GAG Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
41. g any method We recommend using the PureLink HQ Mini Plasmid Purification Kit or the PureLink HiPure Plasmid Miniprep Kit see page 34 Refer to www invitrogen com or contact Technical Support for more information on a large selection of plasmid purification columns Note that because you are purifying a vector that acts as a low copy number plasmid you may need to increase the amount of bacterial culture that you use to prepare your plasmid construct pBAD202 D lacZ is included in the kit as an expression control This control plasmid contains directionally TOPO Cloned gene encoding B galactosidase see page 32 for a map Transform 10 ng of the control plasmid into One Shot TOP10 cells using the procedure on page 13 or page 14 Once you have some clones that you wish to characterize we recommend the following strategy to determine the optimal expression level 1 Pilot Expression Vary the amount of arabinose over a 10 000 fold range 0 00002 to 0 2 to determine the approximate amount of arabinose needed for maximum expression of your protein See next page for protocol 2 To optimize expression of your protein try arabinose concentrations spanning the amount determined in Step 1 or perform a time course Note If your protein is insoluble analyze the supernatant and the pellet of lysed cells for expression of soluble protein see page 19 Continued on next page 17 Expressing the PCR Product Continued
42. h J 1991 The FtsQ Protein of Escherichia coli Membrane Topology Abundance and Cell Division Phenotypes Due to Overproduction and Insertion Mutations J Bacteriol 173 2187 2195 Dalbey R E and Wickner W 1985 Leader Peptidase Catalyzes the Release of Exported Proteins from the Outer Surface of the Escherichia coli Plasma Membrane J Biol Chem 260 15925 15931 Deutscher M P 1990 Guide to Protein Purification In Methods in Enzymology Vol 182 J N Abelson and M I Simon eds Academic Press San Diego CA Guzman L M Barondess J J and Beckwith J 1992 FtsL an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli J Bacteriol 174 7716 7728 Guzman L M Belin D Carson M J and Beckwith J 1995 Tight Regulation Modulation and High Level Expression by Vectors Containing the Arabinose Ppa p Promoter J Bacteriol 177 4121 4130 Holmgren A 1985 Thioredoxin Ann Rev Biochem 54 237 271 Innis M A Gelfand D H Sninsky J J and White T S 1990 PCR Protocols A Guide to Methods and Applications Academic Press San Diego CA Katti S K LeMaster D M and Eklund H 1990 Crystal Structure of Thioredoxin from E coli at 1 68 Angstroms Resolution J Mol Biol 212 167 184 Kuhn A and Wickner W 1985 Isolation of Mutants in M13 Coat Protein That Affect its Synthesis Processing and Assembly into Phage J Biol Chem 260
43. he amplification conditions based on the size of your insert and the sequence of your insert specific primer If you are using this technique for the first time we recommend performing restriction analysis in parallel Artifacts may be obtained because of mispriming or contaminating template The protocol below is provided for your convenience Other protocols are also suitable Materials Needed PCR SuperMix High Fidelity see page 34 Appropriate forward and reverse PCR primers 20 uM each Procedure 1 For each sample aliquot 48 uL of PCR SuperMix High Fidelity into a 0 5 mL microcentrifuge tube Add 1 uL each of the forward and reverse PCR primer 2 Pick5 colonies and resuspend them individually in 50 uL of the PCR cocktail from Step 1 above Incubate reaction for 10 minutes at 94 C to lyse cells and inactivate nucleases Amplify for 20 to 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C NP Ho Analyze by agarose gel electrophoresis If you have problems obtaining transformants or the correct insert perform the control reactions described on pages 28 29 These reactions will help you troubleshoot your experiment After you have identified the correct clone purify the colony and make a glycerol stock for long term storage We recommend that you also store a stock of plasmid DNA at 20 C 1 Streak the original colony out for single colony on LB plates containing 50 uL mL kanamy
44. invitrogen com or contact Technical Support page 36 Expressing your protein with the N terminal thioredoxin and or the C terminal peptide tags increases the size of your recombinant protein The table below lists the expected increase in the molecular weight of your recombinant fusion protein Be sure to account for any additional amino acids between the tag and your fusion protein Peptide Tag Expected Size Increase kDa N terminal Thioredoxin 13 kDa C terminal V5 6xHis 3 kDa Continued on next page Analyzing Samples Continued Optimizing Expression Expressing Toxic Proteins Purifying Recombinant Fusion Proteins Removing the N terminal Leader by Enterokinase After you have detected expression of your protein of interest perform some experiments to further optimize expression e Use the Pilot Expression protocol on page 18 but vary the arabinose concentration over a smaller range For example if you obtained the best expression at 0 002 arabinose in the medium try 0 0004 0 0008 0 001 0 004 and 0 008 e You may also perform a time course of induction to determine if varying the time increases expression Take time points every hour over a 5 to 6 hour period e If your protein is insoluble analyze the supernatant and pellet of lysed cells when you vary the arabinose concentration see Preparing Samples for Soluble Insoluble Protein page 19 e Store your cell lysates at 20 C
45. ion 213 251 Arabinose minimal promoter 248 276 Ribosome binding site 329 332 His Patch Thioredoxin ORF 346 674 TrxFus Forward priming site 655 672 Enterokinase recognition site 691 705 TOPO recognition site 1 718 722 Overhang 723 726 TOPO recognition site 2 727 731 V5 epitope 748 789 Polyhistidine 6xHis region 799 816 pBAD Reverse priming site 872 889 rrnB transcriptional termination region 922 1079 Kanamycin resistance gene c 1448 2242 pUC origin 2340 3013 araC ORF c 3544 4422 c complementary strand Continued on next page 30 Map and Features of pBAD202 D TOPO Continued Features of The pBAD202 D TOPO vector contains the following elements All features pBAD202 have been functionally tested D TOPO Feature Benefit araBAD promoter Ppap Provides tight dose dependent regulation of heterologous gene expression Guzman et al 1995 O region Binding site of AraC that represses transcription from Psp O region Binding site of AraC that represses transcription of the araC promoter Pc transcribed on the opposite strand CAP binding site Site where CAP cAMP binding protein binds to activate transcription from Pran and Pc I and L regions Binding sites of AraC that activate transcription from Paap 10 and 35 regions Binding sites of RNA polymerase for transcription from Paap Ribosome binding site Increases efficiency of recombinant fusion pro
46. k the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 250 uL of room temperature S O C medium to the tubes Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour oO g75g Fw 7 Spread 100 200 pL from each transformation on a prewarmed selective plate and incubate overnight at 37 C We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies 8 An efficient TOPO Cloning reaction may produce several hundred colonies Pick 5 colonies for analysis see Analyzing Positive Clones page 15 Continued on next page 13 Transforming One Shot TOP10 Competent Cells Continued Transformation by Use ONLY electrocompetent cells for electroporation to avoid arcing Do not use the One Shot TOP10 chemically competent cells for electroporation Electroporation NN NEN 7 X Y o i E 1 Add 3 uL of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 11 into a 0 1 cm cuvette containing 50 uL of electrocompetent E coli and mix gently Do not mix by pipetting up and down Avoid formation of bubbles Electroporate your samples using your own protocol and your electroporator Note If you have problems with arcing see below Immediately add 250 uL of room temperature S O C medium to the cuvette Transfer the solution to a 15 mL snap cap tube e g Falcon and shake for at le
47. keep the agarose melted Transform 2 to 4 pL directly into TOP10 One Shot cells using the method on page 13 Note Cloning efficiency may decrease with purification of the PCR product To produce a single band optimize your PCR conditions 27 Performing the Control Reactions Introduction We recommend performing the following control TOPO Cloning reactions the first time you use the kit to help you evaluate your results Performing the control reactions involves producing a control PCR product using the reagents included in the kit and using this product directly in TOPO Cloning reaction Before Starting For each transformation prepare two LB plates containing 50 ug mL kanamycin see page 24 for recipe Producing the Use your thermostable proofreading polymerase and the appropriate buffer to Control PCR amplify the control PCR product Follow the manufacturer s recommendations Product for the polymerase you are using 1 To produce the 750 bp control PCR product set up the following 50 uL PCR Control DNA Template 10 ng 1uL 10X PCR Buffer appropriate for enzyme 5 pL dNTP Mix 0 5 uL Control PCR Primers 0 1 ug uL 1 pL Sterile Water 41 5 pL Thermostable polymerase 1 2 5 unit uL 1 uL Total Volume 50 uL 2 Amplify using the following cycling parameters Step Time Temperature Cycles Initial Denaturation 2 minutes 94 C 1X Denaturation 1 minute 94 C Annealing 1 minute 55 C 25X Ext
48. mple Use the stock solutions prepared in Step 6 and add arabinose to the five 9 mL cultures as follows Note For the positive and negative controls it is not necessary to test all concentrations of arabinose Use only the highest concentration of arabinose Tube Stock Solution Volume mL Final Concentration 1 0 00296 0 09 0 00002 2 0 02 0 09 0 0002 3 0 2 0 09 0 002 4 2 0 09 0 02 5 20 0 09 0 2 10 Grow at 37 C with shaking for 4 hours 11 Take 1 mL samples at 4 hours and treat as in Step 7 and 8 You will have a total of ten samples for each transformant and two samples for each control Proceed to Analyzing Samples next page Analyzing Samples Preparing Samples Preparing Samples for Soluble Insoluble Protein Polyacrylamide Gel Electrophoresis Before starting prepare SDS PAGE gels or use one of the pre cast polyacrylamide gels available from Invitrogen see below to analyze all the samples you have collected Note To analyze your samples for soluble protein see the next section 1 When all the samples have been collected from Steps 8 and 11 on page 18 resuspend each cell pellet in 80 uL of 1X SDS PAGE sample buffer Boil 5 minutes and centrifuge briefly Load 5 10 pL of each sample on an SDS PAGE gel and electrophorese Save your samples by storing them at 20 C 1 Thaw and resuspend each pellet in 500 uL of Lysis Buffer see page 25 for recipe 2 Freez
49. o One Shot TOP10 cells is generally much lower when compared to the number of colonies obtained after transforming other TOPO vectors e g pCR T7 TOPO e Directional TOPO Cloning generally yields 2 to 5 fold fewer colonies than traditional bidirectional TOPO TA Cloning e Transforming low copy number TOPO plasmids generally yields 2 to 5 fold fewer colonies than transforming high copy number TOPO plasmids To compensate for the lower transformation efficiency using the pBAD202 D TOPO e Increase the amount of TOPO Cloning reaction that you transform into TOP10 cells use 3 pL e Increase the amount of transformed cells that you plate use 100 200 uL for chemically competent cells and 50 100 uL for electrocompetent cells Example When directionally TOPO Cloning a 750 bp test insert into the pBAD202 D TOPO vector we generally obtain 500 1 500 total colonies Although fewer total colonies are obtained greater than 90 of the colonies contain plasmid with your PCR insert in the correct orientation 1 Add 3 uL of the TOPO Cloning reaction from Performing the TOPO Cloning Reaction Step 2 page 11 into a vial of One Shot TOP10 Chemically Competent E coli and mix gently Do not mix by pipetting up and down 2 Incubate on ice for 5 to 30 minutes Note Longer incubations on ice seem to have a minimal effect on transformation efficiency The length of the incubation is at the user s discretion Heat shoc
50. ote Because the pBAD202 D TOPO vector acts as a low copy number plasmid you may need to increase the amount of bacterial culture to obtain enough plasmid DNA for sequencing or analysis purposes Use extra care during purification to obtain plasmid DNA of sufficiently pure quality for sequencing see below Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert You may sequence your construct to confirm that your gene is in frame with the N terminal His Patch thioredoxin peptide and the C terminal V5 epitope and polyhistidine 6xHis tag The TrxFus Forward and pBAD Reverse primers are included in the kit to help you sequence your insert Refer to the diagram on page 8 for the location of the primer binding sites If you download the sequence from www invitrogen com note that the overhang sequence GTGG is shown already hybridized to CACC No DNA sequence analysis program allows us to show the overhang without the complementary sequence Continued on next page 15 Analyzing Transformants Continued Analyzing Transformants by PCR Important Long Term Storage You may analyze positive transformants using PCR For PCR primers use a combination of the TrxFus Forward primer or the pBAD Reverse primer and a primer that hybridizes within your insert Determine t
51. product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 or outlicensing lifetech com This product is licensed under U S Patent Nos 5 284 933 and 5 310
52. sequence CACC at the 5 end Make sure that the reverse PCR primer does not contain the sequence CACC at the 5 end Used Taq polymerase or a Taq proofreading polymerase mixture for PCR Use a proofreading polymerase for PCR Long PCR product Increase the incubation time of the TOPO reaction from 5 minutes to 30 minutes Gel purify the PCR product to remove primer dimers and other artifacts PCR reaction contains artifacts i e does not run as a single discrete band on an agarose gel Optimize your PCR using the proofreading polymerase of choice Gel purify your PCR product to remove primer dimers and smaller PCR products Cloning large pool of PCR products or a toxic gene Increase the incubation time of the TOPO reaction from 5 minutes to 30 minutes Use a0 5 1 to 2 1 molar ratio of PCR product TOPO vector 22 Continued on next page Troubleshooting Continued TOPO Cloning Reaction and Transformation continued Problem Reason Solution Large percentage of inserts cloned in the incorrect orientation Incorrect PCR primer design Make sure that the forward PCR primer contains the sequence CACC at the 5 end Reverse PCR primer is complementary to the GTGG overhang at the 5 end Make sure that the reverse PCR primer does not contain the sequence CACC at the 5 end Large number of incorrect inserts cloned PCR reaction cont
53. sign Important Below is the sequence of the C terminus of a theoretical protein The stop codon is underlined GCG GTT AAG TCG GAG CAC TCG ACG ACT GCA TAG 3 To fuse the ORF in frame with a C terminal tag remove the stop codon by starting with nucleotides homologous to the last codon TGC and continue upstream The reverse primer will be 5 TGC AGT CGT CGA GTG CTC CGA CTT 3 This will amplify the C terminus without the stop codon and allow you to join the ORF in frame with a C terminal tag If you do not want to join the ORF in frame with a C terminal tag simply design the reverse primer to include the stop codon 5 CTA TGC AGT CGT CGA GTG CTC CGA CTT 3 Remember that the pBAD202 D TOPO vector accepts blunt end PCR products Refer to the diagram of the TOPO Cloning site on page 8 to help you design your primers When synthesizing PCR primers do not add 5 phosphates to the primers because 5 phosphates prevent the synthesized PCR product from ligating into the pBAD202 D TOPO vector We recommend that you gel purify your oligonucleotides especially if they are long gt 30 nucleotides Continued on next page Designing PCR Primers Continued TOPO Cloning Site Use the diagram below to design suitable PCR primers to clone and express your PCR product in pBAD202 D TOPO Restriction sites are labeled to indicate the actual cleavage site The vector sequence of pBAD202 D TOPO is available
54. tein expression HP thioredoxin Provides a highly efficient fusion partner for translation of the fusion protein TrxFus forward priming site Allows sequencing of the insert in the sense orientation Enterokinase recognition site Asp Asp Asp Asp Lys Allows removal of the N terminal tag from the recombinant fusion protein using an enterokinase such as EKMax TOPO Cloning site directional Allows rapid cloning of your PCR product for expression in E coli C terminal V5 epitope tag Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of the recombinant fusion protein by the Anti V5 Antibodies Southern et al 1991 C terminal 6xHis tag Allows purification of the recombinant fusion protein on metal chelating resins e g ProBond Allows detection of the recombinant fusion protein with the Anti His C term Antibodies Lindner et al 1997 rrnB transcription termination region Strong transcription termination region Kanamycin resistance gene Allows selection of the plasmid in E coli pUC origin Allows low copy replication and growth in E coli araC gene Encodes the regulatory protein for tight regulation of the Paap promoter Lee 1980 Schleif 1992 31 Map of pBAD202 D lacZ Description pBAD202 D lacZ 7 520 bp is a control vector that contains the gene for B galactosidase The lacZ gene was amplified an
55. tion If you have quantitated the yield of your PCR product you may need to adjust the concentration of your PCR product before proceeding to TOPO Cloning Tip For the pBAD202 D TOPO vector using 1 5 ng of a 1 kb PCR product or 5 10 ng of a 2 kb PCR product in a TOPO Cloning reaction generally results in a suitable number of colonies 100 50 Relative Activity colonies reaction 0 0 1 1 10 PCR Product Vector Molar Ratio Continued on next page Performing the TOPO Cloning Reaction Continued Using Salt Solution in the TOPO Cloning Reaction Performing the TOPO Cloning Reaction Perform TOPO Cloning in a reaction buffer containing salt i e using the stock salt solution provided in the kit Note that the amount of salt added to the TOPO Cloning reaction varies depending on whether you plan to transform chemically competent cells provided or electrocompetent cells see page 34 for ordering information e Ifyou are transforming chemically competent E coli use the stock Salt Solution as supplied and set up the TOPO Cloning reaction as directed below e Ifyou are transforming electrocompetent E coli reduce the amount of salt in the TOPO Cloning reaction to 50 mM NaCl 2 5 mM MgCl to prevent arcing during electroporation Dilute the stock Salt Solution 4 fold with water to prepare a 300 mM NaCl 15 mM MgCl Dilute Salt Solution Use the Dilute Salt Solution to set up the TOPO Cloning
56. ugh Place the column back into the Wash Tube 9 Add 700 uL Wash Buffer W9 with ethanol add 96 100 ethanol to the Wash Buffer according to instructions on the label of the bottle to the column and incubate at room temperature for 5 minutes Centrifuge at 212 000 x g for 1 minute Discard flow through 10 Centrifuge the column at 712 000 x g for 1 minute to remove any residual buffer Place the column into a 1 5 mL Recovery Tube 11 Add 50 uL warm 65 70 C TE Buffer TE to the center of the cartridge Incubate at room temperature for 1 minute 12 Centrifuge at 212 000 x g for 2 minutes The Recovery Tube contains the purified DNA Store DNA at 20 C Discard the column 13 Use 4 uL of the purified DNA for the TOPO Cloning reaction Continued on next page Purifying the PCR Products Continued Low Melt Agarose Note that gel purification will dilute your PCR product Use only chemically Method competent cells for transformation 1 Electrophorese as much as possible of your PCR reaction on a low melt agarose gel 0 8 to 1 2 in TAE buffer Visualize the band of interest and excise the band Place the gel slice in a microcentrifuge tube and incubate the tube at 65 C until the gel slice melts Place the tube at 37 C to keep the agarose melted Use 4 uL of the melted agarose containing your PCR product in the TOPO Cloning reaction page 10 Incubate the TOPO Cloning reaction at 37 C for 5 to 10 minutes to
57. ureLink HQ Mini Plasmid Purification 100 preps K2100 01 Kit PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 100 preps K2100 03 EKMax Enterokinase 250 units E180 01 EK Away Resin 7 5 mL R180 01 Ampicillin Sodium Salt irradiated 200 mg 11593 027 B Gal Antiserum 50 uL R901 25 B Gal Assay Kit 100 reactions K1455 01 B Gal Staining Kit 1 kit K1465 01 X gal 100 mg 15520 034 Invitrogen offers a variety of proofreading thermostable DNA polymerases for generating blunt end PCR products Ordering information is provided below Item Amount Cat no Platinum Pfx DNA Polymerase 100 units 11708 013 AccuPrime Pfx DNA Polymerase 200 reactions 12344 024 Pfx50 DNA Polymerase 100 reactions 12355 012 TOP10 cells are also available as electrocompetent cells See the table below for ordering information Kit Reactions Cat no One Shot TOP10 Electrocomp E coli 10 C4040 50 20 C4040 52 TOP10 Electrocomp Kits 20 C664 55 40 C664 11 120 C664 24 Continued on next page Accessory Products Continued Detecting Recombinant Protein Purifying Recombinant Protein Expression of your recombinant fusion protein can be detected using Anti Thio Anti V5 or Anti His C term antibodies available from Invitrogen Horseradish peroxidase HRP or alkaline phosphatase AP conjugated antibodies allow one step detection using chemiluminescent or colorim
58. usive 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 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Use of the pBAD Directional TOPO Expression Kit is covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the
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