Gateway

• 1. Surender Kumar 2009BS41D

2. Contents Introduction to the Gateway System Defining Gateway technology. Advantages of Gateway cloning. How to generate an entry clone Ways to enter the Gateway system. Gene Expression How to obtain a Gateway expression clone. Multisite Gateway System Cloning multiple fragments into a single vector. Plant Cloning Gateway Vectors. 3. Introduction to the Gateway System To study the structure and function of genes, cloning of thosegenes into appropriate expression vectors is often required. Characterization of genes Subcloning into one or more specialized vectors Restriction enzyme digestions Several approaches: Homologous recombination in Escherichia coli or Yeast Site-specific transposition Limitation: specific hosts selection schemes 4. Recombinational Cloning The phage recombinationbasedinvitroconservative DNA segments flanked by recombination sites A new vector also containing recombination sites Bacteriophage integrase recombination proteinssite-specific 5. Phage lambda recombination in E. coli cosPhageattPThe Gateway System relies on232 bpspecific and non cross-reacting att sequencesx attB E. coli 21 bpBP ClonaseIntegration (Int, IHF)Excision (Int, IHF, Xis)LR ClonaseattLattR96 bp157 bpLysogenThe specificity is given by the 7 nucleotides of the core region 6. att site A defined length of DNA that constitutes a recombination site. There are 4 classes of att sites called attB, attP, attL, and attR. ccdB gene A counterselectable gene that allows for negative selection of unwanted byproduct plasmids after recombination.Donor (pDONR) Vector A vector with attP sites flanking a counterselectable gene that recombines with a gene of interest flanked by attB sites. BP reaction A recombination event between attB and attP sites catalyzed by BP Clonase II Entry (pENTR) clone A vector that contains gene of interest flanked by attL or attR sites. LR reaction A recombination event between attL and attR sites catalyzed by LR Clonase IIDestination (DEST) Vector An application-geared vector with attR sites flanking a counterselectable gene that will recombine with one or more entry clones. 7. The Gateway ReactionsccdB attL1attL2Entry CloneattR1+ccdB attR2LR clonaseDestination VectorattB1attB2Expression CloneattP1+attP2Donor VectorBP clonase KanRAmpRAmpRKanR 8. The Gateway Cloning System Your Source ORF collectionGene synthesis GeneGeneGeneProtein LocalizationMaintains reading frameNo restriction enzymesLibraryDirectional cloningNo ligation1 hour, roomtemperature reaction with >99% efficiencyNo re-sequencingPCRCompatible with automationReversible reactionsYour Application Gene Entry CloneGeneGeneProtein PurificationRNAiGeneGeneCell-FreeProtein interactionGene1Gene2Gene3Gene4Your Application 9. Key Benefits of the Gateway Technology Efficiently and easily shuttle insert DNA from one expression plasmidto another Simplify the cloning workflow and save time Utilize Ultimate ORF clones, a pre-made Gateway collection Simultaneously clone, in a specific order and orientation, up to 4 DNAfragments into one plasmid, Yielding a high proportion of desired clones. In vitro reactions eliminates problems of plasmid segregation Sequence alterations to the subcloned DNA segment are not expected. 10. How to generate an entry clone Options for entering the Gateway system BP Clonase reaction LR Clonase reaction Ultimate ORF collection Vector NTI Advance software for in silico cloning 11. Different ways to generate the entry clone P1ccdBL1P2Donor VectorB1+GeneB2+TOPO-Activated Entry VectorattB PCR Product1.L2Gene PCR ProductBP Cloning2. BP ClonaseTOPO CloningTOPOL1GeneL2Entry Clone Ligase3.4. Pre-made entry clone 5. Custom-made entry cloneRestriction/Ligase Cloning L1L1L2digested Entry Vector+B1Genedigested DNA FragmentORFL2ORF Collection 12. 1. BP Cloning The Reaction gene attL1 ccdB attP1 attB1geneattB2+Entry CloneattP2 attR1Donor Vector KanRattL2ccdBattR2+KanRBP Clonase90-99% correct clones on Kan plates 13. 2. TOPO Cloning -TOPOTA 14. 3. Restriction/Ligase cloningUse when there are convenient sites to cut insert out of another plasmid Must cut out ccdB gene by using one of four RE sites flanking the ccdB Reading frame of insert must be considered, as well as downstream expression elements 15. 4. Pre-existing ORF collection Invitrogens Ultimate ORF collection16,272 human ORFs (Oct 2006 release) Amber stop codons Sequence verified Ready to use in LR reactionshttp://orf.invitrogen.com/cgi-bin/ORF_Browser 16. 5. Custom Gene Synthesis Quick and cost-effective No PCR amplification necessary 100% accuracy (sequence verified) Optional codon optimization for expression 17. In silico cloning using Vector NTI AdvanceTM 11.5 DNA of interestPrimers for PCR reactionCloning Strategy 18. Gene Expression Destination vectors Effects of att sites on prokaryotic and mammalian expression How to create your own destination vector. 19. Obtaining a Gateway Expression Clone geneattB1ccdBgene attL1attR1attL2 Entry Clone KanRccdB+attR2attP1AmpRLR Clonase IIExpression CloneattP2 Donor VectorDestination VectorattB2+AmpRKanR90-99% correct clones on Amp plates 20. Choice of the expression system Cell-freeEasy of useCost of media and EquipmentPos-translational Modifications (Probability of protein function)Time RequirementBacteriaYeastInsectMammalian 21. Destination vectors for protein expression E. coliNative protein expressionCell free Mammalian InsectN-terminal fusion protein expressionYeastVirusC-terminal fusion protein expression 22. Examples of Destination vectors For the expression of C-terminal fusion or native proteinsFor expression of N-terminal fusion proteins 23. Methods for Obtaining Gateway Expression Clones attR1ccdBattR2attL1+Destination VectorGene Entry CloneLR ReactionattB1GeneattB2Expression CloneTOPO Reaction attB1attB2TOPO Expression vector+Gene PCR ProductattL2 24. Transfer of the CAT Gene into Multiple Destination Vectors Destination Vector/ApplicationColoniesBackgroundAnalysis*Native15,00004/46xHis-fusion10,65004/4GST-fusion9,20004/4Thioredoxin-fusion11,00004/4Sequencing + Strand13,95004/4Sequencing - Strand8,95004/4Native protein7,800154/46xHis6,350304/4CMV-promoter7,95004/4SFV4,50004/4Tet-regulated promoter6,35004/4E. coliInsectMammalian* positives/total 25. Effect of att sites on prokaryotic protein expressionpET300/NT-DestpET302 NT-HisMM = MagicMedia LB = Luria-Bertani medium + 1mM IPTG 26. Effect of att sites on mammalian protein expression 123 1) pCDNA3.1/lacZ/V5His CMVplacZV5HisLacZ 2) pDEST40/lacZ/V5His CMVpattB1attB2lacZV5His3) MultiSite CMV/lacZ/V5His attB4Extracts normalized for total protein Western blot probed with anti V5 antibodyCMVpattB1lacZattB2V5HisattB3 27. Gateway Conversion Kit R2R1Gateway Cassette+R1destination vectorPromoterlinearized vectorConversion cassettes can be used with any vector or system, even the proprietary onesR2 28. Multisite Gateway System How to clone up to 4 DNA fragments simultaneously into onedestination vector. Expression of multiple genes in HeLa cells. Testing the effects of promoters and regulatory elements onprotein expression. 29. ` 30. More att sequences neededStandard GatewayCTGCTTTTTTGTACAAACTTGattB1CAGCTTTCTTGTACAAAGTTGattB2CAACTTTATTATACAAAGTTGattB3CAACTTTTCTATACAAAGTTGattB4CAACTTTTGTATACAAAGTTGattB5MultiSite Gateway 31. 2-fragment MultiSite Gateway Pro PCR Fragments pDONRsattB1attB5rattB5attB2XXXXattP1attP5rattP5attP2BP reactions attL5Entry ClonesattL2X attL1attR5X Destination VectorsattR1attR2LR reaction Expression clonesattB1attB5attB2 32. 3-fragment MultiSite Gateway Pro PCR FragmentsattB4attB4rattB3rattB3attB2X pDONRsattB1XXXXXattP1attP4attP4rattP3rattP3attP2BP reactionsattR1attL3XattR4Destination vectorattL1attL4XEntry clonesattR3CmRccdBattL2attR2LR reaction Expression cloneattB1attB4attB3attB2 33. 4-fragment MultiSite Gateway Pro PCR FragmentsattB1X pDONRsattP1attB5rattB5attB3rXXXattP5attP5rattB4rXXattB4attP4attP4rattP3rattB3attB2XXattP3attP2BP reactions attL5Entry ClonesattL4attL3X attL1XXattR5attR4attL2attR3X Destination VectorsattR1attR2LR reaction Expression clonesattB1attB5attB4attB3attB2 34. MultiSite Gateway Three-Fragment Vector Construction Kit PCR FragmentsattB1rattB1attB2attB2rattB3X pDONRsattB4XXXXXattP4attP1rattP1attP2attP2rattP3BP reactionsattR4attR2XattL1Destination vectorattL4attR1XEntry clonesattL2CmRccdBattL3attR3LR reaction Expression cloneattB4attB1attB2attB3 35. Typical Results Number of recombining fragmentsExpected # colonies per 10 L reactionTypical recombination efficiency (%)110 3 -10690 -100210 3 -10580 -100310 3 -10470 -90410 2 -10330 -80 36. Shortcomings when co-transfecting two plasmids EGFPmRFPPCAGEGFP mRFPEGFPPlasmid 1 mRFPPlasmid 2 37. Expression of Multiple Genes in Human Cells CFPApCMVB1B1 pCMV B5BB4 pEF1YFPYFPB3B4 pEF1CFPB3CFPB2B2YFP 38. Testing of Expression Elements using MultiSite Gateway Kozak or Promoter IRESEGFPpABGHHeLaaurora A cdc 2 cyclin B1 cyclin E CMV EF1-a ( CAG ) ( SV40 )Kozak or Gtx 2xGtx 5xGtx 12xGtx EMCV mHCV2a mHCV33 mHCV45 HCV2a HCV33 HCV45Determination of expression level of EGFPIRES ( Internal Ribosome Entry Site ) 39. Kozak orPromoter IRESEGFPpAHeLaTranscriptional signals with Kozak40.035035.03002930.0250150 10010.0505.013 1315.09 11477mHCV4520.0mHCV3325.0 200mHCV2aEMCV12xGtx5xGtx2xGtxGtxKozakEF1-aCMVcyclin Ecyclin B1cdc 2None0.00aurora ARelative activityTranslational signals with CMV promoter 40. Applications Optimized multigene delivery without co-transfection Expression of enzymatic pathways Expression of multi-subunit protein complexes Gene knock-down Variablegene expression levels using different expressionelements Combinatorial tagging 41. Summary for MultiSite Gateway Technology MultiSite Gateway Three-Fragment Vector Construction Kit Compatible withUltimate ORF clonesMultiSite Gateway ProattL1-attL2 entry clonesMultiSite Gateway Pro entry clonesattR4-attR3 DEST vectorsattR1-attR2 DEST vectorsAvailable forOnly 3-fragment cloning2-, 3-, or 4-fragment cloningApplicationsVector constructionVector constructionPromoter analysisPromoter analysis Expression of multiple genes in one plasmid Reporter analysis and more 42. PLANT CLONING The Gateway cloning provides expression platform of choice forplant systems, with a wide range of vectors for different plant species and purposes. AdaptedGateway vectors for Agrobacterium-mediated transformation, silencing studies, and many other applications have been cited in over 150 peer-reviewed articles. 43. Many of these vectors have been made available from academicand commercial entities By Invitrogen. Some of these are listed here: Department of Plant Systems Biology, University of Ghent pEarly gate vectors Tag protein expression in plants Arabidopsis Information Resource InPlanta Innovation Inc. 44. MAPK signalling cascades extracellular stimuli such as environmental stresses and pathogens Transgenic rice plants Overexpressed OsMAPK33 suppressed OsMAPK33 45. Vector construction and plant transformation Gateway binary vector pB7WG2D and pB7GWIWG2(II) were used foroverexpression (top) and suppression (bottom)of OsMAPK33 With increased salinity no difference in salt tolerance between OsMAPK33-suppressed linesand their wild-type plants. the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+ /Na+ ratio inside the cell. suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. 46. Conclusion Gateway cloning eliminates the disadvantages of restriction enzymebased cloning It offers expression possibilities that have been impractical orinvolve many cumbersome steps with traditional restriction enzyme cloning. Manipulating large numbers of Genes was not possible in a uniformmannerindependent of size, sequence, or restriction sites. Gateway Cloning is nearly independent of these constraints and ishighly efficient Genes may be cloned, subcloned, screened for phenotypes, andretrieved from procedures.screeningprotocolswithhigh-throughput 47. Thank you