User Manual E. coli Expression System with Gateway ® Technology
Contents
1. Prepare plasmid DNA using your method of choice We recommend using the S N A P MidiPrep Kit Cat no K1910 01 or the PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 for isolation of plasmid DNA Note that since you are purifying a low copy number plasmid you should increase the amount of bacterial culture used to prepare your plasmid construct For most purposes ampicillin works well for selection of transformants and expression experiments However if you find that your expression level is low you may want to use carbenicillin instead The resistance gene for ampicillin encodes a protein called p lactamase This protein is secreted into the medium where it hydrolyzes ampicillin inactivating the antibiotic Since p lactamase is catalytic ampicillin is rapidly removed from the medium resulting in non selective conditions If your plasmid is unstable this may result in the loss of plasmid and low expression levels Carbenicillin is generally more stable than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help to increase expression levels by preventing loss of the pDEST expression plasmid If you wish to use carbenicillin perform your transformation and expression experiments in LB containing 50 ug ml carbenicillin Note If your gene is highly toxic increasing the concentration of carbenicillin used from 50 ug ml to 200 ug ml may help to increase expression lev2. The figure below summarizes the features of the pENTR ous vector The complete sequence for pENTR ous is available from our website www invitrogen com or by contacting Technical Support see page 37 attL1 bases 99 198 complementary strand gus gene bases 228 2039 attL2 bases 2041 2140 pUC origin bases 2200 2873 C Kanamycin resistance gene bases 2990 3805 C C complementary strand 35 Recipes Lysis Buffer 36 50 mM potassium phosphate pH 7 8 400 mM NaCl 100 mM KCI 10 glycerol 0 5 Triton X 100 10 mM imidazole 1 Prepare 1M stock solutions of KH PO and K HPO 2 For100 ml dissolve the following reagents in 90 ml of deionized water 4 7 ml KHPO 2 3 g NaCl 0 75 g KCl 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 at 4 C Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporat
3. e The presence of the N terminal or C terminal GST tag in pDEST 15 and pDEST 4 respectively allows purification of recombinant fusion protein using glutathione agarose Refer to the manufacturer s instructions to purify your protein 27 Troubleshooting Expression Introduction No Expression Low Expression Due to Plasmid Instability Low Expression Due to Toxicity Precautions 28 Use the information below to troubleshoot your expression experiment Sequence your construct and make sure it is in frame with the N terminal or C terminal tag as appropriate If you are using ampicillin for selection in your expression experiments and see low levels of expression you may be experiencing plasmid instability due to the absence of selective conditions This occurs as the ampicillin is destroyed by B lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism You may want to substitute carbenicillin for ampicillin in your transformation and expression experiments see page 23 for more information TM When expressing recombinant proteins in the BL21 AI strain one can generally assume that the recombinant protein is toxic to bacterial cells when any of the following occurs e No transformants are obtained after following the BL21 AI One Shot Transformation Procedure page 24 or a combination of large and small irregular colonies appears on the plate e The initial cu
4. glucose in addition to 0 2 arabinose Appendix Regulation by L Arabinose Introduction Regulation of the araBAD Paap Promoter Glucose Repression The L arabinose regulatory circuit is briefly described below The araBAD promoter Pgap used to control expression of T7 RNA polymerase in BL21 AT is both positively 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 L arabinose the AraC dimer contacts the O2 and I half sites of the araBAD operon forming a 210 bp DNA loop see figure below For maximum transcriptional activation two events are required e L Arabinose binds to AraC and causes the protein to release the O site and bind the Iz site that 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 Io AraC dimer No transcription arabinose Transcription Basal expression levels can be repressed by introducing glucose to the growth medium Glucose acts by lowering cAMP levels which in turn decreases the binding of CAP As cAMP levels are lowered transcriptional activation is decreased 29 Map and Features of the pDEST Vectors pDEST 14 Map The map below shows the elements of pDEST 14 DNA from the ent
5. 4093 pBR322 origin bases 4238 4911 ROP ORF bases 5282 5473 C C complementary strand continued on next page 3l Map and Features of the pDEST Vectors continued pDEST 17 Map The map below shows the elements of pDEST 17 DNA from the entry clone replaces the region between bases 147 and 1830 The complete sequence for pDEST 17 is available from our website www invitrogen com or by contacting Technical Support see page 37 E ES ATG 6xHis Lang Cm ccdB attR2 Comments for pDEST 17 6354 nucleotides T7 promoter bases 21 40 Ribosome binding site RBS bases 86 92 Initiation ATG bases 101 103 6xHis tag bases 113 130 attR1 bases 140 264 Chloramphenicol resistance gene Cm bases 373 1032 ccdB gene bases 1374 1679 attR2 bases 1720 1844 T7 transcription termination region bases 1855 1983 bla promoter bases 2471 2569 Ampicillin bla resistance gene bases 2570 3430 pBR322 origin bases 3575 4248 ROP ORF bases 4619 4810 C C complementary strand continued on next page 32 Map and Features of the pDEST Vectors continued pDEST 24 Map The map below shows the elements of pDEST 24 DNA from the entry clone replaces the region between bases 78 and 1761 The complete sequence for pDEST 24 is available from our website www invitrogen com or by contacting Technical Support see page 37 attR1 Cm ccdB attR2 GST Comments for pDEST 24 6961 nucleotides T7 promoter bas
6. Lys Leu Pro Glu Met Leu Lys 432 GCA TAT AGT AAA GAC TTT GAA ACT CTC AAA GTT GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG AAA Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe 498 ATG TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT GAT CAT GTA ACC CAT CCT GAC TTC Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys 564 ATG TTG TAT GAC GCT CTT GAT GTT GTT TTA TAC ATG GAC CCA ATG TGC CTG GAT GCG TTC CCA AAA Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys 630 TTA GTT TGT TTT AAA AAA CGT ATT GAA GCT ATC CCA CAA ATT GAT AAG TAC TTG AAA TCC AGC AAG Tyr Ile Ala Trp Pro Leu Glin Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser 696 TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC ACG TTT GGT GGT GGC GAC CAT CCT CCA AAA TCG 799 l Asp Leu Val Pro Arg Pro Trp Ser Asn Gln Thr ser Leu Tyr Lys LVS Ala GUY sar crs eao ee 762 GAT CTG GTT CCG CGT CCA TGG TCG AAT CAA ACA AGT TIG TAC AAA AAA GCA GGC TNN SS ene CTA GAC CAA GGC GCA GGT ACC AGC TTA GTT TGT TCA AAC ATG TTT Geen 2482 l attB1 NACCCAGCTT TCTTGTACAA AGTGGTTTGA TTCGACCCGG GATCCGGCTG CTAACAAAGC CCGAAAGGAA NTGGGTCGAA AGAACATGTT TCACCAAACT L attB2 continued on next page 16 Creating an Expression Clone continued Recombination Region of pDEST 17 20 100 Features of the Recombination Region The r
7. Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala 1942 CTT CCT TAT TAT ATT GAT GGT GAT GTT AAA TTA ACA CAG TCT ATG GCC ATC ATA CGT TAT ATA GCT Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala 2008 GAC AAG CAC AAC ATG TTG GGT GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA ATG CTT GAA GGA GCG Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val 2074 GTT TTG GAT ATT AGA TAC GGT GTT TCG AGA ATT GCA TAT AGT AAA GAC TTT GAA ACT CTC AAA GTT Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr 2140 GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG AAA ATG TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr 2206 TTA AAT GGT GAT CAT GTA ACC CAT CCT GAC TTC ATG TTG TAT GAC GCT CTT GAT GTT GTT TTA TAC Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile 2272 ATG GAC CCA ATG TGC CTG GAT GCG TTC CCA AAA TTA GTT TGT TTT AAA AAA CGT ATT GAA GCT ATC Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 2338 CCA CAA ATT GAT AAG TAC TTG AAA TCC AGC AAG TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC 1 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Pro Trp Gly Ser Gly Cys 2404 ACG TTT GGT GGT GGC GAC CAT CCT CCA AAA TCG GAT CTG GTT CCG CGT CCA TGG GGA TCC GGC TGC EEN 2470 TAA CAA
8. 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 37 Purchaser Notification Introduction Information for European Customers Limited Use Label License No 19 Gateway Cloning Products 38 Use of the E coli Expression System with Gateway Technology is covered under the licenses detailed below TM The BL21 AT E co
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10. ng DNA in a volume of 1 5 ul into each vial of BL21 AT One Shot cells and mix by tapping gently Do not mix cells by pipetting up and down Incubate on ice for 30 minutes Heat shock 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 Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 30 minutes Spread 20 ul and 100 ul from each transformation on a prewarmed selective plate and incubate overnight at 37 C We generally plate 2 different volumes to ensure that at least 1 plate has well spaced colonies Select a transformant and proceed to Pilot Expression next page Note Expression can vary between clones You may wish to characterize additional transformants continued on next page Expressing Your Recombinant Protein Materials Needed Pilot Expression LB media containing 100 ug ml ampicillin or 50 ug ml carbenicillin 37 C shaking incubator 20 L arabinose supplied with the kit Box 4 20 glucose if needed prepare in sterile deionized water Lysis Buffer see page 36 for a recipe Liquid nitrogen 1X and 2X SDS PAGE sample buffer see page 36 for a recipe Reagents and apparatus for SDS PAGE gel see the next page Boiling water bath Sterile water Pick 3 or 4 transformants from BL21 AI One Shot Transformation Procedure Step 8 page 24 and culture them in 5 ml of LB medium containing 100 u
11. tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour Spread 20 ul and 100 ul from each transformation on a prewarmed selective plate and incubate overnight at 37 C We generally plate 2 different volumes to ensure that at least 1 plate has well spaced colonies An efficient LR recombination reaction should produce hundreds of colonies gt 5000 colonies if the entire LR reaction is transformed and plated 21 Analyzing Transformants Analyzing Positive 1 Pick 5 colonies and culture them overnight in LB or SOB medium containing Clones Analyzing Transformants by PCR Confirming the Expression Clone Sequencing 22 100 ug ml ampicillin 2 Isolate plasmid DNA using your method of choice We recommend using the S N A P MidiPrep Kit Cat no K1910 01 or the PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 available from Invitrogen Note Since pDEST 14 pDEST 15 pDEST 17 and pDEST 24 are low copy number plasmids 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 3 Analyze plasmids by restriction analysis to confirm the presence of the insert You may also analyze positive transformants using PCR For PCR primers use a primer that hybridizes within the vector e g I7 Promoter
12. use these cells to propagate or maintain your expression clone Genotype F ompT hsdSg eme gal dcm araB T7RNAP tetA TM The BL21 AT strain is an E coli B r strain and does not contain the lon protease It is also deficient in the outer membrane protease OmpT The lack of these proteases reduces degradation of heterologous proteins expressed in this strain The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase T7 RNAP gene in the araB locus allowing expression of the T7 RNAP to be regulated by the araBAD promoter see page 29 for more information The presence of the tet A gene confers resistance to tetracycline and permits verification of strain identity using tetracycline Accessory Products Introduction The products listed in this section may be used with the E coli Expression System with Gateway Technology For more information refer to www invitrogen com or call Technical Support see page 37 Additional Many of the reagents supplied in the E coli Expression System with Gateway Products Technology as well as other products suitable for use with the kit are available separately from Invitrogen Ordering information for these reagents is provided below Item Quantity Catalog no LR Clonase II Enzyme Mix 20 reactions 11791 020 100 reactions 11791 100 Library Efficiency DH5a Competent Cells 5 x 0 2 ml 18263 012 BL
13. 11 11819 018 Once you have selected an entry vector refer to the manual for the specific entry vector you are using for instructions to construct an entry clone All entry vector manuals are available for downloading from our website or by contacting Technical Support see page 37 Your gene of interest in the entry clone must e Contain an ATG initiation codon and a Shine Dalgarno sequence RBS with optimal spacing for proper translation initiation in E coli Shine and Dalgarno 1975 Note If you clone your gene of interest into an entry vector that supplies a Shine Dalgarno RBS e g PENTR SD D TOPO or pENTR 11 then your gene of interest need only include an ATG initiation codon e Contain a stop codon TM Refer to the diagram of the recombination region of pDEST 14 on page 15 to help you design a strategy to generate your entry clone continued on next page Generating an Entry Clone continued Points to Consider Before Recombining into pDEST 15 and pDEST 17 Points to Consider Before Recombining into pDEST 24 pDEST 15 and pDEST 17 are N terminal fusion vectors and contain an ATG initiation codon upstream of the GST and 6 His tags respectively In each vector a Shine Dalgarno RBS is included upstream of the initiation ATG to ensure optimal spacing for proper translation initiation in E coli Your gene of interest in the entry clone must e Bein frame with the N terminal tag after reco
14. 21 AI One Shot Chemically Competent 20 x 50 ul C6070 03 E coli Gateway pDEST 14 Vector 20 ug 11801 016 Gateway pDEST 15 Vector 20 ug 11802 014 Gateway pDEST 17 Vector 20 ug 11803 012 Gateway pDEST 24 Vector 20 ug 12216 016 Ampicillin Sodium Salt irradiated 200 mg 11593 027 Carbenicillin Disodium Salt 5g 10177 012 Purification of The presence of the polyhistidine 6xHis tag in pDEST 17 allows purification of Recombinant your recombinant fusion protein using a nickel charged agarose resin such as Protein ProBond or Ni NTA Ordering information is provided below Item Quantity Catalog no ProBond Nickel Chelating Resin 50 ml R801 01 150 ml R801 15 ProBond Purification System 6 purifications K850 01 Ni NTA Agarose 10 ml R901 01 25 ml R901 15 100 ml R901 10 Ni NTA Purification System 6 purifications K950 01 Overview Introduction The pET Expression System Features of the Vectors Introduction The E coli Expression System with Gateway Technology contains a series of Gateway adapted destination vectors designed to facilitate high level inducible expression of recombinant proteins in E coli using the pET system Depending on the vector chosen the pDEST vectors allow production of native N terminal or C terminal tagged recombinant proteins see table below Vector Fusion Peptide Fusion Tag pDEST 14 pDEST 15 N terminal Glutathione S transferase GST
15. AGCCC GAAAGGAAGC TGAGTTGGCT GCTGCCACCG CTGAGCAATA 18 Performing the LR Recombination Reaction Introduction Positive Control LR Clonase II Enzyme Mix Materials Needed Once you have produced an entry clone containing your gene of interest you are ready to perform an LR recombination reaction between the entry clone and the appropriate pDEST vector and to transform the reaction mixture into Library Efficiency DH5a to select for an expression clone It is important to have everything you need set up and ready to use to ensure that you obtain the best results We suggest that you read this section and the one entitled Transforming Library Efficiency DH5a Cells page 21 before beginning We also recommend that you include a positive control see below and a negative control no LR Clonase in your experiment The pENTR gus plasmid is included in the E coli Expression System with Gateway Technology for use as a positive control for LR recombination and expression Use of the pENTR gus entry clone in an LR recombination reaction with a pDEST vector will allow you to generate an expression clone containing the gene encoding B glucuronidase gus LR Clonase II enzyme mix is supplied with the kit Cat no 11824 026 only or available separately from Invitrogen to catalyze the LR recombination reaction The LR Clonase II enzyme mix combines the proprietary enzyme formulation and 5X LR Clonase Reac
16. Gateway Technology LR Recombination Reaction The Basis of T7 Regulated Expression The Gateway Technology is a universal cloning method that takes advantage of the site specific recombination properties of bacteriophage lambda Landy 1989 to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems To express your gene of interest in E coli using the Gateway Technology simply 1 Clone your gene of interest into a Gateway entry vector of choice to create an entry clone 2 Perform an LR recombination reaction between the entry clone and a Gateway destination vector e g pDEST 14 pDEST 15 pDEST 17 pDEST 24 Transform Library Efficiency DH5a E coli and select for an expression clone TM Purify plasmid and transform your expression construct into BL21 AI Induce expression of your recombinant protein with L arabinose For more detailed information about Gateway Technology refer to the Gateway Technology with Clonase II manual To generate an entry clone refer to the manual for the entry vector you are using The Gateway Technology with Clonase II manual and entry vector manuals are available for downloading from our website www invitrogen com or by contacting Technical Support see page 37 You will perform an LR recombination reaction between the entry clone and your destination vector of choice to generate an expression clone The LR recombination r
17. Invitrogen by technologies E coli Expression System with Gateway Technology Gateway adapted destination vectors for cloning and high level expression of native or tagged recombinant proteins in E coli Catalog nos 11824 026 11801 016 11802 014 11803 012 12216 016 Rev Date 7 June 2010 Manual part no 25 0517 MAN0000278 Table of Contents Table of ontentss oe ee eun e eh es AE REDE e es T S M D Ae o ets 3 Kit Contents and Ee 4 Accessory Productsuon atii dte tard eee edd ed deiude dta iei 7 Introduction RE 8 Overview ee eegene Li oret edite Meek Sy 8 The B2 I AT BOE EE 10 Experimental Outline enee Eeer 11 INTIS sae E 12 Generating an Entry Elo ie dete eee ede edi its a lo ihc aan Seon gn Sheba da ees deeg 12 Creating am Expression ClIon e teu etel thc e ined retirer rae pe bre aee checks detenta terns 14 Performing the LR Recombination Reaction sse netten 19 Transforming Library Efficiency DH e 21 Analyzing Transformants EEN ER General Guidelines for Expression EEN 23 Transforming BEL21 AT One Shot Cells auto onte ei nni ne deat b cda edet tite 24 Expressing Your Recombinant Protein EEN 25 Troubleshooting EXpressiORt 4 red appetite od edite e ant davkes dena ade i neiges 28 ell quim M 29 Regulation by L Arabimnoee sse trennen a Tea E E e ETER net 29 Map and Features of the pDEST Vectors adeo tented e e epe 30 Map of CR RE 35 RECIPES ssi ede
18. Primer Invitrogen Cat no N560 02 and one that hybridizes within your insert You will have to determine the amplification conditions If you are using this technique for the first time you may want to perform 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 suitable Materials Needed PCR SuperMix High Fidelity Invitrogen Cat no 10790 020 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 SuperMix containing primers remember to make a patch plate to preserve the colonies for further analysis 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 oggetto Visualize by agarose gel electrophoresis The ccdB gene mutates at a very low frequency resulting in a very low number of false positives True expression clones will be ampicillin resistant and chloram phenicol sensitive Transformants containing a plasmid with a mutated ccdB gene will be ampicillin and chloramphenicol resistant To check your putative expression clone t
19. Smith et al 1986 pDEST 17 N terminal 6xHis pDEST 24 C terminal Glutathione S transferase GST Smith et al 1986 For more information about the Gateway Technology see the next page The pET system was originally developed by Studier and colleagues and takes advantage of the high activity and specificity of the bacteriophage T7 RNA polymerase to allow regulated expression of heterologous genes in E coli from the T7 promoter Rosenberg et al 1987 Studier and Moffatt 1986 Studier et al 1990 For more information about T7 regulated expression see the next page pDEST 14 pDEST 15 pDEST 17 and pDEST 24 contain the following elements e T7 promoter for high level T7 RNA polymerase regulated expression of the gene of interest in E coli Studier and Moffatt 1986 Studier et al 1990 e N or C terminal fusion tags for detection and purification of recombinant fusion proteins choice of tag depends on the particular vector see above e Two recombination sites att R1 and attR2 downstream of the T7 promoter for recombinational cloning of the gene of interest from an entry clone e Chloramphenicol resistance gene Cm located between the two attR sites for counterselection e The ccdB gene located between the attR sites for negative selection e Ampicillin resistance gene for selection in E coli e pBR322 origin for low copy replication and maintenance of the plasmid in E coli Overview continued The
20. Studier F W 1987 Vectors for Selective Expression of Cloned DNAs by T7 RNA Polymerase Gene 56 125 135 Schleif R S 1992 DNA Looping Ann Rev Biochem 61 199 223 Shine J and Dalgarno L 1975 Terminal Sequence Analysis of Bacterial Ribosomal RNA Correlation Between the 3 Terminal Polypyrimidine Sequence of 16 5 RNA and Translational Specificity of the Ribosome Eur J Biochem 57 221 230 Smith D B Davern K M Board P G Tiu W U Garcia E G and Mitchell G F 1986 Mr 26 000 Antigen of Schistosoma japonicum Recognized by Resistant WEHI 129 Mice is a Parasite Glutathione S transferase Proc Natl Acad Sci USA 83 8703 8707 Studier F W and Moffatt B A 1986 Use of Bacteriophage T7 RNA Polymerase to Direct Selective High Level Expression of Cloned Genes J Mol Biol 189 113 130 Studier F W Rosenberg A H Dunn J J and Dubendorff J W 1990 Use of T7 RNA Polymerase to Direct Expression of Cloned Genes Meth Enzymol 185 60 89 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 41 Notes 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
21. ample Positive Control Entry clone 50 150 ng reaction 1 7 ul Destination vector 150 ng ul 1 ul 1 ul pENTR gus 50 ng ul 2 ul TE Buffer pH 8 0 to 8 ul 5 ul Remove the LR Clonase II Enzyme Mix from 20 C and thaw on ice 2 minutes Vortex the LR Clonase II Enzyme Mix briefly twice 2 seconds each time To each sample above add 2 ul of LR Clonase II Enzyme Mix Mix well by pipetting up and down Reminder Return LR Clonase II Enzyme Mix to 20 C immediately after use Incubate reactions at 25 C for 1 hour Note For most applications 1 hour will yield a sufficient number of colonies for analysis Depending on your needs the length of the recombination reaction can be extended up to 18 hours For large plasmids 2 10 kb longer incubation times will yield more colonies Add 1 ul of Proteinase K solution to each reaction Incubate for 10 minutes at 37 C Proceed to Transforming Library Efficiency DH5a TM Cells next page Note You may store the LR reaction at 20 C for up to 1 week before transformation if desired Transforming Library Efficiency DH5a Cells Introduction Materials Needed Note Transformation Protocol Once you have performed the LR recombination reaction you will transform competent E coli Library Efficiency DH5a chemically competent E coli Box 3 are included with the E coli Expression System to facilitate transformation LR recombinatio
22. by mixing the 14 20 entry clone and the appropriate pDEST vector with Gateway LR Clonase II Enzyme Mix 3 Transform the recombination reaction into competent 21 Library Efficiency DH5a cells and select for expression clones 4 Analyze transformants for the presence of insert by 22 restriction enzyme digestion or colony PCR 5 Optional Sequence to confirm that the gene of interest 22 is cloned in frame with the appropriate N terminal or C terminal tag 6 Prepare purified plasmid DNA of the expression clone 23 24 and transform into BL21 AI One Shot cells 7 Picka transformant and perform a pilot expression 25 26 study Add L arabinose to induce expression of your recombinant protein 8 Purify your recombinant protein if desired 27 11 Methods Generating an Entry Clone Introduction Points to Consider Before Recombining into DDEST 14 12 To recombine your gene of interest into pDEST 14 pDEST 15 pDEST 17 or pDEST 24 you will need an entry clone containing the gene of interest Many entry vectors are available from Invitrogen to facilitate generation of entry clones see table below For more information about each vector see our website or contact Technical Support see page 37 Entry Vector Catalog no pENTR D TOPO K2400 20 pENTR SD D TOPO K2420 20 pENTR 1A 11813 011 pENTR 2B 11816 014 pENTR 3C 11817 012 pENTR 4 11818 010 pENTR
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24. e use of this technology including the enclosed materials based upon the following conditions 1 these materials are to be used for non commercial research purposes only A separate license under patents owned by BSA is required for any commercial use including the use of these materials for research purposes or production purposes by any commercial entity Information about commercial license may be obtained from The Office of Technology Transfer Brookhaven National Laboratory Bldg 475D P O Box 5000 Upton New York 11973 5000 Phone 516 344 7134 2 No materials that contain the cloned copy of the T7 gene 1 the gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this sub license and agrees to be bound by its terms This limitation applies to strains BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE CE6 BL21 SI Competent Cells and any derivatives that are made of them You may refuse this sub license by returning this product unused in which case Life Technologies accept return of the product with a full refund By keeping or using this product you agree to be bound by the terms of this license This product is the subject of one or more of U S Patent Numbers 5 910 438 6 180 407 and 7 176 029 and corresponding foreign patents and is sold under license from the Universit Libre de Bruxelles for research purposes only ccdB selection technology is desc
25. eaction is mediated by LR Clonase II Enzyme Mix a mixture of the bacteriophage A Integrase Int and Excisionase Xis proteins and the E coli Integration Host Factor IHF protein For more information about the LR recombination reaction see the Gateway Technology with Clonase II manual The pET expression system uses elements from bacteriophage T7 to control expression of heterologous genes in E coli In the pDEST 14 pDEST 15 pDEST 17 and pDEST 24 vectors expression of the gene of interest is controlled by a strong bacteriophage 17 promoter In bacteriophage 17 the T7 promoter drives expression of gene 10 10 T7 RNA polymerase specifically recognizes this promoter To express the gene of interest it is necessary to deliver T7 RNA polymerase to the cells by inducing expression of the polymerase or infecting the cell with phage expressing the polymerase In the E coli Expression System with Gateway Technology T7 RNA polymerase is supplied by the BL21 AI host E coli strain in a regulated manner see the next page for more information about the strain The BL21 AI Description of the Strain Regulating Expression of T7 RNA Polymerase 10 E coli Strain The BL21 AT E coli strain is included in the kit and is intended for use as a host for expression of T7 RNA polymerase regulated genes The BL21 AI strain is derived from the BL21 strain Grodberg and Dunn 1988 Studier and Moffatt 1986 and contains a c
26. ecombination region of the expression clone resulting from pDEST 17 x entry clone is shown below e The location of the 6xHis tag is indicated to help you determine if your gene will be in frame with the 6xHis tag after recombination e Shaded regions correspond to those DNA sequences transferred from the entry clone into the pDEST 17 vector by recombination Non shaded regions are derived from the pDEST 17 vector e The underlined nucleotides flanking the shaded region correspond to bases 147 and 1830 respectively of the pDEST 17 vector sequence T7 promoter I TTAATACGAC TCACTATAGG AATTATGCTG AGTGATATCC r transcription start l GAGACCACAA CGGTTTCCCT CTAGAAATAA TTTTGTTTAA CTCTGGTGTT GCCAAAGGGA GATCTTTATT AAAACAAATT 147 6xHis tag I Met Ser Tyr Tyr His T ATG TCG TAC TAC CAT A TAC AGC ATG ATG GTA His His CAC CAT GTG GTA TNN 2 z2 NACCCAGCTT zijn EE NTGGGTCGAA sel L l His His His Leu Glu Ser Thr Ser Leu CAC CAT CAC CTC GAA TCA ACA AGT TTG GTG GTA GTG GAG CTT AGT TGT TCA AAC L Shine Dalgarno aoe ee CTTTAAGAAG GAGATATACA GAAATTCTTC CICTATATGT Tyr Lys Lys Ala Gly TAC AAA AAA GCA GGC ATG TTT GE 1830 attB1 TCTTGTACAA AGTGGTTGAT TCGAGGCTGC TAACAAAGCC CGAAAGGAAG AGAACATGTT TCACCAACTA AGCTCCGACG ATTGTTTCGG GCTTTAATTC attB2 continued on next page 17 Creating an Expression Clone continued Recombination The recombination region of the expression clo
27. els 23 Transforming BL21 Al One Shot Cells Modulating Gene Expression Materials Needed BL21 AI One Shot Transformation Procedure 24 TM To modulate expression of your gene of interest in BL21 AI cells use L arabinose to induce expression of T7 RNA polymerase L arabinose is supplied with the BL21 AI cells but is also available from Sigma Cat no A3256 Glucose to repress basal transcription of T7 RNA polymerase and thereby your gene of interest optional Add to plates and or media to a final concentration of 0 1 glucose if needed TM Purified DNA of your pDEST expression clone 1 10 ng ul BL21 AI One Shot chemically competent cells supplied with the kit Box 4 use one vial per transformation pUC19 control supplied with the kit Box 4 use as a control for transformation if desired S O C Medium supplied with the kit Box 4 warm to room temperature LB plates containing 100 ug ml ampicillin or 50 ug ml carbenicillin 2 plates for each transformation prewarm to 37 C for 30 minutes 37 C incubator shaking and non shaking 42 C water bath Follow the instructions below to transform your expression construct into BL21 AI One Shot cells If you are including the pUC19 control transform 10 pg of DNA You will need one vial of cells per transformation 1 2 DI Qv OL We COS Thaw on ice one vial of BL21 AI One Shot cells per transformation Add 5 10
28. emic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for profit organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee 10 per clone payable to Invitrogen We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes that such clones incorporate the Gateway Technology and that the purchase of Gateway Clonase from Invitrogen is required for carrying out the Gateway recombinational cloning reaction This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerful technology in their research Use of Invitrogen s Gateway Technology including Gateway clones for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen s licensing de
29. es 25 44 attR1 bases 71 195 Chloramphenicol resistance gene Cm bases 304 963 ccdB gene bases 1305 1610 attR2 bases 1651 1775 GST tag bases 1783 2451 T7 transcription termination region bases 2466 2594 bla promoter bases 3082 3180 Ampicillin bla resistance gene bases 3181 4041 pBR322 origin bases 4186 4859 ROP ORF bases 5230 5421 C C complementary strand continued on next page 33 Map and Features of the pDEST Vectors continued Features ofthe pDEST 14 6422 bp pDEST 15 7013 bp pDEST 17 6354 bp and pDEST 24 Vectors 6961 bp contain the following elements All features have been functionally tested Feature Benefit 17 promoter Permits high level IPTG inducible expression of your recombinant protein in E coli strains expressing the T7 RNA polymerase Ribosome binding site i e Shine Dalgarno sequence in pDEST 15 and pDEST 17 only Optimally spaced from the initiation ATG in the N terminal tag for efficient translation of the PCR product N terminal glutathione S transferase GST tag in pDEST 15 only Allows affinity purification of recombinant fusion protein using glutathione agarose N terminal 6xHis tag in pDEST 17 only Permits affinity purification of recombinant fusion protein using a metal chelating resin such as ProBond or Ni NTA attR1 and attR2 sites Bacteriophage A derived DNA recombination sequences that permit recombinational clo
30. est for growth on LB plates containing 30 ug ml chloramphen icol A true expression clone will not grow in the presence of chloramphenicol Optional To confirm that your gene of interest is in frame with the appropriate tags if any you may sequence your expression construct General Guidelines for Expression Introduction BL21 AI Strain Plasmid Preparation Choosing a Selection Agent Using Carbenicillin BL21 AI One Shot E coli are included with the E coli Expression System with Gateway Technology Box 4 for use as the host for expression You will need purified plasmid DNA of your pDEST expression construct to transform into BL21 AI Since each recombinant protein has different characteristics that may affect expression we recommend performing a time course of expression to determine the best conditions to express your protein TM The BL21 AI E coli strain is specifically designed for recombinant protein expression from any T7 based expression vector Because T7 RNA polymerase levels can be tightly regulated by L arabinose the BL21 AT strain is especially suited to express genes that may be toxic to other BL21 strains where basal expression of T7 RNA polymerase is leakier Each time you perform an expression experiment you will transform your plasmid into BL21 AI Do not use this strain for propagation and maintenance of your plasmid Use a general cloning strain e g DH5a instead
31. g ml ampicillin or 50 ug ml carbenicillin Grow at 37 C with shaking until the ODs reaches 0 6 to 1 0 Use these cultures to inoculate fresh LB medium containing 100 ug ml ampicillin or 50 ug ml carbenicillin to an OD600 of 0 05 0 1 1 20 dilution of the initial culture This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density Use a volume appropriate for taking time points if desired Grow the cultures until they reach mid log phase OD 0 4 2 to 3 hours Split each culture into two cultures Add L arabinose to a final concentration of 0 296 to one of the cultures You will now have two cultures one induced one uninduced Remove a 500 ul aliquot from each culture centrifuge at maximum speed in a microcentrifuge for 30 seconds and aspirate the supernatant Freeze the cell pellets at 20 C These are the zero time point samples Continue to incubate the cultures at 37 C with shaking Take time points for each culture every hour for 2 to 4 hours For each time point remove 500 ul from the induced and uninduced cultures and process as described in Steps 5 and 6 Proceed to the next section continued on next page 25 Expressing Your Recombinant Protein continued Preparing Samples Preparing Samples for Soluble Insoluble Protein Polyacrylamide Gel Electrophoresis Analyzing Samples 26 Before starting this procedure make sure that you have an a
32. hose DNA sequences transferred from the entry clone into the pDEST 15 vector by recombination Non shaded regions are derived from the pDEST 15 vector e The underlined nucleotides flanking the shaded region correspond to bases 799 and 2482 respectively of the pDEST 15 vector sequence T7 promoter r 3 transcriptional start Shine Dalgarno 1 Ee 21 AAATTAATAC GACTCACTAT AGGGAGACCA CAACGGTTTC CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT Glutathione S transferase Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu 101 ACAT ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA CCC ACT CGA CTT CTT TTG Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn 168 GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG TAT GAG CGC GAT GAA GGT GAT AAA TGG CGA AAC Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu 234 AAA AAG TTT GAA TTG GGT TTG GAG TTT CCC AAT CTT CCT TAT TAT ATT GAT GGT GAT GTT AAA TTA Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys 300 ACA CAG TCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC ATG TTG GGT GGT TGT CCA AAA Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile 366 GAG CGT GCA GAG ATT TCA ATG CTT GAA GGA GCG GTT TTG GAT ATT AGA TAC GGT GTT TCG AGA ATT Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser
33. hromosomal insertion of the gene encoding 17 RNA polymerase T7 RNAP into the araB locus of the araBAD operon placing regulation of the T7 RNAP gene under the control of the araBAD promoter The araB gene is deleted in this strain Because the T7 RNAP gene is inserted into the araB locus of the araBAD operon expression of T7 RNA polymerase can be regulated by the sugars L arabinose and glucose e To induce expression from the araBAD promoter use L arabinose Lee 1980 Lee et al 1987 To modulate expression simply vary the concentration of L arabinose added e _ To repress basal expression from the araBAD promoter use glucose Note In the absence of glucose basal expression from the araBAD promoter is generally low Lee 1980 Lee et al 1987 Adding glucose further represses expression from the araBAD promoter by reducing the levels of 3 5 cyclic AMP Miyada et al 1984 For more information on the mechanism of expression and repression of the ara regulon see the Appendix page 29 or refer to Schleif 1992 Experimental Outline Experimental The table below outlines the steps required to express your gene of interest in Outline E coli from pDEST 14 pDEST 15 pDEST 17 or pDEST 24 Step Action Page 1 Design an appropriate scheme and clone your gene of 12 13 interest into the Gateway entry vector of choice to generate an entry clone 2 Perform an LR recombination reaction
34. ice 80 C E coli Kit 4 BL21 AI One Shot Chemically Dry ice 80 C Competent E coli Kit Note The individual Gateway pDEST vectors Catalog nos 11801 016 11802 014 11803 012 12216 016 are shipped on wet ice Upon receipt store at 20 C continued on next page Kit Contents and Storage continued Destination The following destination vectors Box 1 are supplied with the E coli Expression Vectors System with Gateway Technology Store the vectors at 20 C Note Catalog nos 11801 016 11802 014 11803 012 and 12216 016 contain 40 ul of the appropriate pDEST vector at 150 ng ul concentration in 10 mM Tris HCI 1 mM EDTA pH 8 0 only Reagent Composition Amount pDEST 14 Vector 40 ul of vector at 150 ng ul in TE buffer pH 8 0 20ug pDEST 15 Vector 40 ul of vector at 150 ng ul in TE buffer pH 8 0 20ug pDEST 17 Vector 40 ul of vector at 150 ng pl in TE buffer pH 8 0 20 ug pDEST 24 Vector 40 ul of vector at 150 ng ul in TE buffer pH 8 0 20ug LR Clonase Il The following reagents are included with the Gateway LR Clonase II Enzyme Mix Enzyme Mix Box 2 Store Box 2 at 20 C for up to 6 months For long term store at 80 C Reagent Composition Amount LR Clonase II Enzyme Mix Proprietary 40 ul Proteinase K solution 2 us ul in 40 ul 10 mM Tris HCl pH 7 5 20 mM CaCl 50 glycerol pENTR gus Positive Control 50 ng ul in TE Buffer pH 8 0 20 u
35. its components for Commercial Purposes The buyer may transfer information or materials made through the employment 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 Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Life Technologies under the patents identified above to distribute such materials Transfer of such materials and or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the 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 Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture u
36. l DH5a Competent The Library Efficiency DH5a Competent E coli kit Box 3 includes the following items Transformation efficiency is gt 1 x 10 cfu ug DNA Store Box 3 at 80 C E coli Item Composition Amount Library Efficiency Chemically 5 x 200 ul Competent DH5a S O C Medium 2 tryptone 2 x6 ml may be stored at room 0 5 yeast extract temperature or 4 C 10 mM NaCl 2 5 mM KCI 10 mM MgCl 10 mM MgSO 20 mM glucose pUC19 Control DNA 10 pg ul in 5 mM Tris HCl 50 ul 0 5 mM EDTA pH 8 0 continued on next page Kit Contents and Storage continued Genotype of DH5a BL21 AI One Shot Competent E coli Genotype of BL21 AI Use this strain to propagate and maintain your expression clone Genotype F recAl end A1 hsdR17 rx mi supE44 X thi 1 gyrA96 relA1 The BL21 AI One Shot Chemically Competent E coli Kit Box 4 includes the following items Transformation efficiency is 2 1 x 10 cfu ug DNA Store Box 4 at 80 C Item Composition Amount BL21 AI chemically competent 21 x 50 ul cells S O C Medium 2 tryptone 6 ml may be stored at room 0 5 yeast extract temperature or 4 C 10 mM NaCl 2 5 mM KC 10 mM MgCl 10 mM MgSO 20 mM glucose 20 L arabinose 20 L arabinose in sterile 1ml water pUC19 Control DNA 10 pg ul in 5 mM Tris HCI 50 ul 0 5 mM EDTA pH 8 0 Note Use this strain for expression only Do not
37. li strain is genetically modified and carries a chromosomal insertion of a cassette containing the T7 RNA polymerase T7 RNAP gene As a condition of sale use of 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 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 purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Life Technologies Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Life Technologies Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose The buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or
38. lture does not grow see Step 1 of Pilot Expression page 25 e It takes longer than 5 hours after a 1 20 dilution of the initial culture for the fresh culture to reach an ODeo 0 4 see Steps 2 and 3 of Pilot Expression page 25 e The cells lyse after induction with L arabinose see Step 4 of Pilot Expression page 25 Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest see below These methods all assume that the T7 based expression plasmid has been correctly designed and created e Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase i e DH5a TM e Perform a fresh transformation of BL21 AI cells before each expression experiment e After following the transformation protocol on page 24 plate the transform ation reaction on LB plates containing 100 ug ml ampicillin and 0 1 glucose The presence of glucose represses basal expression of 17 RNA polymerase TM e Following transformation of BL21 AI cells using the protocol on page 24 pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 ug ml ampicillin or 50 ug ml carbenicillin and 0 1 glucose if desired When the culture reaches ODeo 0 4 induce expression of the recombinant protein by adding L arabinose to a final concentration of 0 276 e When performing expression experiments supplement the growth medium with 0 1
39. mbination e Contain a stop codon TM TM Refer to the diagram of the recombination region of pDEST 15 or pDEST 17 on pages 16 and 17 respectively to help you design a strategy to generate your entry clone TM pDEST 24 is a C terminal fusion vector Your gene of interest in the entry clone must e Contain an ATG initiation codon and a Shine Dalgarno RBS with optimal spacing for proper translation initiation in E coli Note If you clone your gene of interest into an entry vector that supplies a Shine Dalgarno RBS e g PENTR SD D TOPO or pENTR 11 then your gene of interest need only include an ATG initiation codon e Beinframe with the C terminal GST tag after recombination e NOT contain a stop codon Refer to the diagram of the recombination region of pDEST 24 on page 18 to help you design a strategy to generate your entry clone 13 Creating an Expression Clone Introduction Experimental Outline Important Propagating the Vectors 14 After you have generated an entry clone you will perform the LR recombination reaction to transfer the gene of interest into the pDEST vector to create your expression clone To ensure that you obtain the best possible results we recommend that you read this section and the next section entitled Performing the LR Recombination Reaction pages 19 20 before beginning To generate an expression clone you will 1 Perform an LR recombination reactio
40. n reaction from Step 7 previous page Library Efficiency DH5a chemically competent cells supplied with the kit Box 3 thaw on ice before use S O C medium supplied with the kit Box 3 warm to room temperature pUC19 control supplied with the kit Box 3 use as a control for transformation if desired LB plates containing 100 ug ml ampicillin two for each transformation warm at 37 C for 30 minutes 42 C water bath 37 C shaking and non shaking incubator Library Efficiency DH5a competent cells are supplied in 5 tubes containing 0 2 ml of competent cells each Each tube contains enough competent cells to perform 4 transformations using 50 ul of cells per transformation Once you have thawed a tube of competent cells discard any unused cells Do not re freeze cells as freezing and thawing of cells will result in the loss of transformation efficiency DU De Oe e For each transformation aliquot 50 ul of Library Efficiency D So competent cells into a sterile microcentrifuge tube Add 1 ul of the LR recombination reaction from Setting Up the LR Recombination Reaction Step 7 previous page into the tube containing 50 ul of Library Efficiency DH5a competent cells and mix gently Do not mix by pipetting up and down Incubate on ice for 30 minutes Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 450 ul of room temperature S O C medium Cap the
41. n using the attL containing entry clone and the attR containing pDEST vector Note Both the entry clone and the destination vector should be supercoiled see Important below Transform the reaction mixture into a suitable E coli host see page 21 Select for expression clones see pages 15 18 for illustrations of the recombination region of expression clones in pDEST 14 pDEST 15 pDEST 17 or pDEST 24 The pDEST 14 pDEST 15 pDEST 17 and pDEST 24 vectors are supplied as supercoiled plasmids Although we have previously recommended using a linearized destination vector for more efficient recombination further testing at Invitrogen has found that linearization of the destination vector is NOT required to obtain optimal results for any downstream application If you wish to propagate and maintain the pDEST 14 pDEST 15 pDEST 17 or pDEST 4 vectors prior to recombination we recommend using 10 ng of the vector to transform One Shot ccdB Survival 2 T1 Chemically Competent Cells Cat no A10460 from Invitrogen The ccdB Survival 2 T1 E coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene To maintain the integrity of the vector select for transformants in media containing 50 100 g ml ampicillin and 15 30 g ml chloramphenicol TM Note Do not use general E coli cloning strains including TOP10 or DH5a for propagation and maintenance as these strains are
42. ne resulting from Region of pDEST 7A x entry clone is shown below pDEST 24 Features of the Recombination Region e The glutathione S transferase GST gene is marked to help you determine if your gene will be in frame with the GST tag after recombination e Shaded regions correspond to those DNA sequences transferred from the entry clone into the pDEST 24 vector by recombination Non shaded regions are derived from the pDEST 24 vector e Theunderlined nucleotides flanking the shaded region correspond to bases 78 and 1761 respectively of the pDEST 24 vector sequence 78 T7 promoter r 2 transcriptional start 1 21 AAATTAATAC GACTCACTAT AGGGAGACCA CAACGGTTTC CCTCTAGATC ACAAGTTTGT ACAAAAAAGC AGGC TNN GGAGATCTCG TGTTCAAACA TGTTTETECG TCCG ANN L J 1761 Glutathione S transferase atm Pro Ala Phe Leu Tyr Lys Val Val Ile Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile eg NAC CCA GCT TTC TTG TAC AAA GTG GTG ATT ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT ZEE NTG GGT CGA AAG AAC ATG TTT CAC CAC TAA TAC L attB2 Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 1810 AAG GGC CTT GTG CAA CCC ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn 1876 TAT GAG CGC GAT GAA GGT GAT AAA TGG CGA AAC AAA AAG TTT GAA TTG GGT TTG GAG TTT CCC AAT Leu Pro Tyr Tyr Ile Asp Gly Asp Val
43. ning of the gene of interest from a Gateway entry clone Landy 1989 Chloramphenicol resistance gene Cm Allows counterselection of the plasmid ccdB gene Permits negative selection of the plasmid C terminal glutathione S transferase GST tag in pDEST 24 only Allows affinity purification of recombinant fusion protein using glutathione agarose T7 transcription termination region Sequence from bacteriophage T7 that permits efficient transcription termination bla promoter Allows expression of the ampicillin resistance gene Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pBR322 origin of replication ori Permits replication and maintenance in E coli ROP ORF Interacts with the pBR322 origin to facilitate low copy replication in E coli Map of pbENTR gus Description Map of Control Vector Comments for pENTR gus 3841 nucleotides pENTR gus is a 3841 bp entry clone containing the Arabidopsis thaliana gene for B glucuronidase gus Kertbundit et al 1991 The gus gene was amplified using PCR primers containing attB recombination sites The amplified PCR product was then used in a BP recombination reaction with pDONR201 to generate the entry clone For more information about the BP recombination reaction refer to the Gateway Technology with Clonase II manual Note The molecular weight of GUS is 68 4 kDa
44. ns dcos a Rita o I fet o i e etie iioii ts tete eid 36 Technical EECH eet et E ee 37 Purchaser Notifications eee ERR Ie Ue e HC ee ee HUE HL Feste iral 38 Gateway Clone Distribution Policy anssen tenen dl coal eod uvae ee 40 EE 41 Kit Contents and Storage Types of Products This manual is supplied with the following products listed below Product Catalog no E coli Expression System with Gateway Technology 11824 026 Gateway pDEST 14 Vector 11801 016 Gateway pDEST 15 Vector 11802 014 Gateway pDEST 17 Vector 11803 012 Gateway pDEST 24 Vector 12216 016 Kit Components contents of each component see pages 5 6 Component Catalog no Each product contains the following components For a detailed description of the 11824 026 11801 016 11802 014 11803 012 12216 016 pDEST 14 Vector J J pDEST 15 Vector pDEST 17 Vector pDEST 24 Vector Gateway LR Clonase II Enzyme Mix E coli Library Efficiency DH5a Competent aal I2 2 Competent E coli BL21 AI One Shot Chemically Y Shipping Storage The E coli Expression System with Gateway Technology is shipped as described below Upon receipt store each item as detailed below Box Item Shipping Storage 1 pDEST Vectors Wet ice 20 C 2 Gateway LR Clonase II Enzyme Mix Dry ice 20 C 3 Library Efficiency DH5a Competent Dry
45. partment at 760 603 7200 References Grodberg J and Dunn J J 1988 ompT Encodes the Escherichia coli Outer Membrane Protease that Cleaves 17 RNA Polymerase During Purification J Bacteriol 170 1245 1253 Kertbundit S Greve H d Deboeck F Montagu M V and Hernalsteens J P 1991 In vivo Random B glucuronidase Gene Fusions in Arabidopsis thaliana Proc Natl Acad Sci USA 88 5212 5216 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 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 arab Activates the araBAD Operon Promoter Proc Natl Acad Sci USA 84 8814 8818 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 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 Rosenberg A H Lade B N Chui D S Lin S W Dunn J J and
46. ppropriate gel for your protein size or use one of the pre cast polyacrylamide gels available from Invitrogen see below and next page If you wish to analyze your samples for soluble protein see the next section 1 When all the samples have been collected from Steps 5 and 7 previous page resuspend each cell pellet in 80 ul of 1X SDS PAGE sample buffer 2 Boil5 minutes and centrifuge briefly Load 5 10 ul 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 Recipes page 36 2 Freeze sample in dry ice or liquid nitrogen and then thaw at 42 C Repeat 2 to 3 times Note To facilitate lysis you may need to add lysozyme 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 electrophore
47. ribed in Bernard et al Positive Selection Vectors Using the F Plasmid ccdB Killer Gene Gene 148 1994 71 74 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 For licensing information for use in other than research please contact Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail This product is the subject of U S Patent No 5 654 176 and or foreign equivalents and is to be used for scientific investigation and research and for no other purpose whatsoever Licenses for commercial use of the above mentioned patents must be negotiated directly with Amrad Corporation 576 Swan Street Richmond Victoria Australia 3121 Telephone 61 3 9208 4000 39 Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions 40 The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing attL1 and attL2 sites may be generated by acad
48. rogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy page 40 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 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 OIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany The GUS positive control vector in these products is claimed in patents and patent applications See U S Patent No 5 599 670 and Great Britain Patent No 2 197 653 licensed to Life Technologies by Cambia Biosystems L L C CBL Use of the GUS gene is restricted to use as a positive control Any other use may require a license from CBL The composition and or use of this product may be claimed in U S Patent No 5 693 489 licensed to Life Technologies Corporation by Brookhaven Science Associates LLC The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U S Department of Energy and is the subject of patents and patent applications assigned to Brookhaven Science Associates LLC BSA By provisions of the Distribution License Agreement granted to Life Technologies covering said patents and patent applications Life Technologies grants you a non exclusive sub license under patents assigned to BSA for th
49. ry clone replaces the region between bases 74 and 1898 The complete sequence for pDEST 14 is available from our website www invitrogen com or by contacting Technical Support see page 37 attR1 Cm ccdB attR2 Comments for pDEST 14 6422 nucleotides T7 promoter bases 21 40 attR1 bases 67 191 Chloramphenicol resistance gene Cm bases 441 1100 ccdB gene bases 1442 1747 attR2 bases 1788 1912 T7 transcription termination region bases 1923 2051 bla promoter bases 2539 2637 Ampicillin bla resistance gene bases 2638 3498 pBR322 origin bases 3643 4316 ROP ORF bases 4687 4878 C C complementary strand continued on next page 30 Map and Features of the pDEST Vectors continued pDEST 15 Map The map below shows the elements of pDEST 15 DNA from the entry clone replaces the region between bases 799 and 2482 The complete sequence for pDEST 15 is available from our website www invitrogen com or by contacting Technical Support see page 37 E ES ATG GST attRi Cm ccdB attR2 Comments for pDEST 15 7013 nucleotides T7 promoter bases 25 44 Ribosome binding site RBS bases 90 96 Initiation ATG bases 105 107 GST tag bases 108 776 attR1 bases 792 916 Chloramphenicol resistance gene CmR bases 1025 1684 ccdB gene bases 2026 2331 attR2 bases 2372 2496 T7 transcription termination region bases 2518 2646 bla promoter bases 3134 3232 Ampicillin bla resistance gene bases 3233
50. se 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 none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein 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 on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 continued on next page Purchaser Notification continued Gateway Clone Distribution Policy Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 23 GUS Control Vector Limited Use Label License No 30 T7 Expression System Limited Use Label License No 54 ULB ccdB Selection Technology Limited Use Label License No 125 GST For additional information about Invit
51. sensitive to CcdB effects continued on next page Creating an Expression Clone continued Recombination The recombination region of the expression clone resulting from Region of pDEST 14 x entry clone is shown below pDEST 14 Features of the Recombination Region e Shaded regions correspond to those DNA sequences transferred from the entry clone into the pDEST 14 vector by recombination Non shaded regions are derived from the pDEST 14 vector e The underlined nucleotides flanking the shaded region correspond to bases 74 and 1898 respectively of the pDEST 14 vector sequence 20 T7 promoter transcription start T TTAATACGAC TCACTATAGG GAGACCACAA CGGTTTCCCT CTAGATCACA AGTTTGTACA AAAAAGCAGG CTNN AATTATGCTG AGTGATATCC CTCTGGTGTT GCCAAAGGGA GATCTAGTGT TCAAACATGT TTTTTCGTCC GANN L attB1 1898 mu rm NACCCAGCTT TCTTGTACAA AGTGGTGATG ATCCGGCTGC TAACAAAGCC CGAAAGGAAG CTGAGTTGGC See SSeS NTGGGTCGAA AGAACATGTT TCACCACTAC TAGGCCGACG ATTGTTTCGG GCTTTCCTTC GACTCAACCG attB2 continued on next page 15 Creating an Expression Clone continued Recombination The recombination region of the expression clone resulting from Region of pDEST 15 x entry clone is shown below pDEST 15 Features of the Recombination Region e The glutathione S transferase GST gene is marked to help you determine if your gene will be in frame with the GST tag after recombination e Shaded regions correspond to t
52. sis apparatus are available from Invitrogen In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits see next page For more information refer to www invitrogen com or call Technical Support see page 37 To determine the success of your expression experiment you may want to perform the following types of analyses 1 Stain the polyacrylamide gel 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 See next page for recommended protein standards and stains 2 Perform a western blot to confirm that the overexpressed band is your desired protein You will need to have an antibody to your protein of interest Note If you are expressing your protein from pDEST 15 or pDEST 24 you may use an antibody to GST to detect your protein continued on next page Expressing Your Recombinant Protein continued Recommended Electrophoresis Accessory Products Note Purifying Recombinant Protein In addition to the pre cast polyacrylamide gel systems Invitrogen offers a wide range of pre mixed buffers protein standards and stains each with its own advantages For more information refer to www invitrogen com or contact Technical Support page 37 Product Quantity Cat no NuPAGE LDS Sample Buffer 4X 10 ml NP0007 250 ml NP0008 No
53. tion Buffer previously supplied as separate components in LR Clonase enzyme mix into an optimized single tube format for easier set up of the LR recombination reaction Use the protocol provided on page 20 to perform the LR recombination reaction using LR Clonase II enzyme mix Note You may perform the LR recombination reaction using LR Clonase enzyme mix if desired To use LR Clonase enzyme mix follow the protocol provided with the product Do not use the protocol for LR Clonase II enzyme mix provided in this manual as reaction conditions differ e Purified plasmid DNA of your entry clone 50 150 ng ul in TE pH 8 0 e pDEST vector Box 1 150 ng pl in TE pH 8 0 e LRClonase II Enzyme Mix Box 2 keep at 20 C until immediately before use e TE Buffer pH 8 0 10 mM Tris HCl pH 8 0 1 mM EDTA e Proteinase K solution supplied with the LR Clonase II Enzyme Mix thaw and keep on ice until use e pENTR gus positive control 50 ng ul in TE pH 8 0 continued on next page 19 Performing the LR Recombination Reaction continued Setting Up the LR Recombination Reaction 20 Follow this procedure to perform the LR recombination reaction between your entry clone and the destination vector If you want to include a negative control set up a separate reaction but omit the LR Clonase II enzyme mix Add the following components to 1 5 ml microcentrifuge tubes at room temperature and mix Component S
54. vex Tris Glycine SDS Sample Buffer 2X 20 ml LC2676 SimplyBlue SafeStain 1L LC6060 SilverQuest Silver Staining Kit 1 kit LC6070 SilverXpress Silver Staining Kit 1 kit LC6100 Colloidal Blue Staining Kit 1 kit LC6025 Novex Sharp Protein Standard Pre stained 2 x 250 ul LC5800 Unstained 2 x 250 ul LC5801 SeeBlue Plus2 Pre Stained Standard 500 ul LC5925 UltraPure Sodium Dodecyl Sulfate SDS 500g 15525 017 Expression of your protein with the N or C terminal tags will increase the size of your recombinant protein The table below lists the increase in the molecular weight of your recombinant fusion protein that you should expect from the tag in each pDEST vector Be sure to account for any additional amino acids between the fusion tag and the start of your protein Vector Fusion Tag Expected Size Increase kDa pDEST 15 N terminal 27 7 pDEST 17 N terminal 2 6 pDEST 24 C terminal 27 9 e The presence of the N terminal 6xHis tag in pDEST 17 allows affinity purification of recombinant fusion protein using a nickel chelating resin such as ProBond or Ni NTA ProBond and Ni NTA resin are available separately from Invitrogen see page 7 for ordering information Refer to the ProBond or Ni NTA manual as appropriate for guidelines to purify your protein Both manuals are available for downloading from our website www invitrogen com or by contacting Technical Support see page 37
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