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A Conduit to Proteomics
Gateway® Technology: A Conduit to Proteomics KDR Biotech Co., Ltd. This is the main title slide – type your name and the title of the presentation in the box.
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The Cloning Bottleneck
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Designing a New Approach to Cloning
Maximize compatibility and flexibility Minimize planning Maintain reading frame Rapid No restriction enzymes No gel purification No ligations High-throughput
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Subcloning an Entry Clone into Multiple Destination Vectors
Gene Yeast 2-Hybrid E. coli Viral Your Vector Mammalian Insect In vitro translation Fusion protein Gene Entry Clone
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Building Entry Clones Insert Three Options Vector Entry Vectors Km
Conventional cloning Directional Gateway® TOPO® Cloning PCR Cloning Vector a tt L1 attL2 Entry Vectors gene Km r MCS at tP1 attP2 Donor Vector ccd B Km r L1 L2 TOPO®-activated Entry Vector Kmr Insert attB PCR Product B2 B1 Restriction fragment PCR Product
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Directional TOPO Cloning®-1
Fast and simple directional PCR cloning strategy with >90% efficiency
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pCR8/GW/TOPO TA Cloning Kit
Directional TOPO Cloning®-2 pCR8/GW/TOPO TA Cloning Kit pCR8/GW/TOPO TA Cloning Kit $ price - One Shot TOP # K - One Shot Mach1-T1R # K TOPO TA Cloning Kit + Gateway entry clone • Direct cloning with PCR product !! • Sequencing vector!! • Gateway Entry vector!!
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pENTR/TEV/D-TOPO Cloning Kit
Directional TOPO Cloning®-4 pENTR/TEV/D-TOPO Cloning Kit •With One Shot TOP10 Chemically Competent E.coli Cat# K •With One Shot Mach1-Tl Chemically Compentent E.coli Cat# K TEV/Drectional TOPO Cloning with Gateway • Efficient cleavage of any N-terminal tag • No need to check orientation! • Gateway Entry vector!!
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Vectors for Gateway® BP Reaction LR Reaction GOI GOI Entry Donor Clone
tP1 attP2 Donor Vector ccd B Km r GOI a tt L1 attL2 Entry Clone gene Km r GOI attB1 attB2 BP Reaction LR Reaction attB 1 2 Expression Clone gene Ap r GOI att R1 R2 Destination Vector ccdB Ap r
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Modification of Recombination
Creation of Direction Specific Selection BP reaction attR1 attR2 attL1 attL2 x ccdB KanR ccdB KanR attB1 attB2 attP1 attP2 x attP1 attP2 attB1 attB2 x LR reaction AmpR ccdB ccdB attL1 attL2 x attR1 attR2 KanR AmpR KanR ccdB: A gene encodes a protein that interferes with E. coli DNA gyrase.
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Gateway PCR Cloning Highly efficient and HTP amenable with recombination method
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Primer Design for att B PCR
Add the att B1 sequence to the 5’-primer Add the att B2 sequence to the 3’-primer att B1 5’ - GGGG ACA AGT TTG TAC AAA AAA GCA GGC TNN NNN... att B2 5’ - GGGG AC CAC TTT GTA CAA GAA AGC TGG GTN NNN... Gene Specific Primer Sequence
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Invitrogen New Product
Gateway BP Clonase II 1. Cat# and Price 이전제품 최근 제품 Gateway BP Clonase Gateway BP Clonase II # (20rxn) ,000원 # (20rxn) ,000원 # (100rxn) 1,807,000원 # (100rxn) ,000원 2. Contents BP Clonase BP Clonase II Gateway BP Clonase Enzyme Mix 80ul 5X BP Clonase Reaction buffer ul 2ug/ul Proteinase K sol ul 30% PEG 8000/30mM Mgcl2 sol ml 50ng/ul Pexp7-tet Positive Control 20ul Gateway BP Clonase II Enzyme Mix 40ul 2ug/ul Proteinase K sol ul 30% PEG 8000/30mM Mgcl2 sol ml 50ng/ul Pexp7-tet Positive Control ul
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attB-PCR product (15~150ng) 1-7 ml pDONR vector 1 ml
BP Protocol attB-PCR product (15~150ng) ml pDONR vector ml TE Buffer, pH to 8 ml 1. Add 2 ml of BP Clonase™ II Enzyme Mix 2. Incubate for 1 hour at 25oC. 3. Add Proteinase K solution and incubate for 10 min at 37oC. 4. Transform competent E. coli
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Cloning PCR Products a DNA mini-prep analysis 1 pUC = 108 CFU/ml
2 After overnight incubation
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Gateway® Technology: Transfer Entry Clones into Expression Clones
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Invitrogen New Product
Gateway LR Clonase II 1. Cat# and Price 이전제품 최근 제품 Gateway LR Clonase Gateway LR Clonase II # (20rxn) ,000원 # (20rxn) 207,000원 # (100rxn) 1,807,000원 # (100rxn) 874,000원 2. Contents (20rxn기준) LR Clonase LR Clonase II Gateway LR Clonase Enzyme Mix 80ul 5X LR Clonase Reaction buffer ul 2ug/ul Proteinase K sol ul 50ng/ul Pentr-gus Positive Control 20ul Gateway LR Clonase Enzyme Mix 40ul 2ug/ul Proteinase K sol ul 50ng/ul Pentr-gus Positive Control 20ul
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Destination Vector (150ng/ml) 1 ml TE Buffer, pH 8.0 to 8 ml
LR Protocol Entry Clone (50~150ng) ml Destination Vector (150ng/ml) ml TE Buffer, pH to 8 ml 1. Add 2 ml of LR Clonase™ II Enzyme Mix 2. Incubate for 1 hour at 25oC. 3. Add Proteinase K solution and incubate for 10 min at 37oC. 4. Transform competent E. coli
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Destination Expression Systems
Native protein expression N-terminal fusion protein expression C-terminal fusion protein expression In vitro E. coli Mammalian Insect Yeast Viral Others
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Parallel Transfer of CAT Gene into Multiple Destination Vectors
Expression Vector Colonies Background Analysis Native (E. coli) 15, /4 6xHis-fusion 10, /4 GST-fusion 9, /4 Thioredoxin-fusion 11, /4 Native protein (baculo) 7, /4 6xHis (baculo) 6, /4 CMV-promoter 7, /4 Tet-regulated promoter 6, /4
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Gateway® reactions with Clonase™ II enzymes
BP Clonase™ II reaction attB-PCR product or linearized expression clone (~ ng) l pDONR™ vector (150 ng) l TE Buffer, pH to 8 l Vortex BP Clonase™ II and add 2 l to above Incubate at 25C for 1 hour Incubate in 1 l Proteinase K at 37 C for 10 min Transform LR Clonase™ II reaction Entry clone (~ ng) l Destination vector (150 ng) 1 l TE Buffer, pH to 8 l Vortex LR Clonase™ II and add 2 l to above Incubate at 25C for 1 hour Incubate in 1 l Proteinase K at 37 C for 10 min Transform Clonase™ II reactions are 10 l volumes instead of 20 l
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Gateway® Systems Applications
Drug discovery , Drug discovery assays
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GATEWAY Collaborations for Building Source Clones
Harvard Institute of Proteomics (Harlow and LaBaer) FLEX: full-length human ORFS and S. cerevisiae Dana Farber Cancer Institute (Vidal) Full-length C. elegans, two-hybrid mapping of C. elegans proteome German Genome Consortium (Korn and Wiemann) Novel full-length human ORFS, two-hybrid mapping of human proteome, high throughput protein localization U.C. Berkeley, LBL, Case Western Reserve University, Curagen Full-length Drosophila Japanese Genome Projects Millennium Project (Sugamo): full-length human ORFs University of Tokyo (Yoshida): full-length S. pombe ORFs Others
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Benefits of Gateway® Technology
Saves time, no need to plan additional cloning strategies Eliminates gene re-amplification after initial cloning entry clone can be archived for future use Reduces sequencing, no need to verify expression clones Provides optimized expression systems
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