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.

The Cloning Bottleneck

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

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

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

Directional TOPO Cloning®-1 Fast and simple directional PCR cloning strategy with >90% efficiency

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 TOP10 # K2500-20 - One Shot Mach1-T1R # K2520-20 TOPO TA Cloning Kit + Gateway entry clone • Direct cloning with PCR product !! • Sequencing vector!! • Gateway Entry vector!!

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# K2525-20 •With One Shot Mach1-Tl Chemically Compentent E.coli Cat# K2535-20 TEV/Drectional TOPO Cloning with Gateway • Efficient cleavage of any N-terminal tag • No need to check orientation! • Gateway Entry vector!!

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

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.

Gateway PCR Cloning Highly efficient and HTP amenable with recombination method

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

Invitrogen New Product Gateway BP Clonase II 1. Cat# and Price 이전제품 최근 제품 Gateway BP Clonase Gateway BP Clonase II #11789-013 (20rxn) 427,000원 #11789-020 (20rxn) 185,000원 #11789-021 (100rxn) 1,807,000원 #11789-100 (100rxn) 824,000원 2. Contents BP Clonase BP Clonase II Gateway BP Clonase Enzyme Mix 80ul 5X BP Clonase Reaction buffer 200ul 2ug/ul Proteinase K sol. 40ul 30% PEG 8000/30mM Mgcl2 sol. 1ml 50ng/ul Pexp7-tet Positive Control 20ul Gateway BP Clonase II Enzyme Mix 40ul 2ug/ul Proteinase K sol. 40ul 30% PEG 8000/30mM Mgcl2 sol. 1ml 50ng/ul Pexp7-tet Positive Control 20ul

attB-PCR product (15~150ng) 1-7 ml pDONR vector 1 ml BP Protocol attB-PCR product (15~150ng) 1-7 ml pDONR vector 1 ml TE Buffer, pH 8.0 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

Cloning PCR Products a DNA mini-prep analysis 1 pUC = 108 CFU/ml 2 After overnight incubation

Gateway® Technology: Transfer Entry Clones into Expression Clones

Invitrogen New Product Gateway LR Clonase II 1. Cat# and Price 이전제품 최근 제품 Gateway LR Clonase Gateway LR Clonase II #11791-019 (20rxn) 427,000원 #11791-020 (20rxn) 207,000원 #11791-043 (100rxn) 1,807,000원 #11791-100 (100rxn) 874,000원 2. Contents (20rxn기준) LR Clonase LR Clonase II Gateway LR Clonase Enzyme Mix 80ul 5X LR Clonase Reaction buffer 200ul 2ug/ul Proteinase K sol. 40ul 50ng/ul Pentr-gus Positive Control 20ul Gateway LR Clonase Enzyme Mix 40ul 2ug/ul Proteinase K sol. 40ul 50ng/ul Pentr-gus Positive Control 20ul

Destination Vector (150ng/ml) 1 ml TE Buffer, pH 8.0 to 8 ml LR Protocol Entry Clone (50~150ng) 1-7 ml Destination Vector (150ng/ml) 1 ml TE Buffer, pH 8.0 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

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

Parallel Transfer of CAT Gene into Multiple Destination Vectors Expression Vector Colonies Background Analysis Native (E. coli) 15,000 0 4/4 6xHis-fusion 10,650 0 4/4 GST-fusion 9,200 0 4/4 Thioredoxin-fusion 11,000 0 4/4 Native protein (baculo) 7,800 15 4/4 6xHis (baculo) 6,350 30 4/4 CMV-promoter 7,950 0 4/4 Tet-regulated promoter 6,350 0 4/4

Gateway® reactions with Clonase™ II enzymes BP Clonase™ II reaction attB-PCR product or linearized expression clone (~15-150 ng) 1-7 l pDONR™ vector (150 ng) 1 l TE Buffer, pH 8.0 to 8 l Vortex BP Clonase™ II and add 2 l to above Incubate at 25C for 1 hour Incubate in 1 l Proteinase K at 37 C for 10 min Transform LR Clonase™ II reaction Entry clone (~50-150 ng) 1-7 l Destination vector (150 ng) 1 l TE Buffer, pH 8.0 to 8 l Vortex LR Clonase™ II and add 2 l to above Incubate at 25C 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

Gateway® Systems Applications Drug discovery , Drug discovery assays

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

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