Gateway Technology A Conduit to Proteomics KDR Biotech

Gateway® Technology: A Conduit to Proteomics KDR Biotech Co. , Ltd.

The Cloning Bottleneck 2

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 3

Subcloning an Entry Clone into Multiple Destination Vectors Gene In vitro translation Fusion protein Gene Viral Gene E. coli Entry Clone Your Vector Gene Yeast 2 -Hybrid Gene Mammalian Insect 4

Building Entry Clones Three Options Conventional cloning Directional TOPO® Cloning Gateway® PCR Cloning Vector ccd. B 2 t. P 2 t. L at t. P 1 L 2 at gene L 1 at at t. L 1 MCS Entry Vectors TOPO®-activated Entry Vector Donor Vector Kmr Kmr Insert Restriction fragment B 1 PCR Product B 2 att. B PCR Product 5

Directional TOPO Cloning®-1 Fast and simple directional PCR cloning strategy with >90% efficiency 6

Directional TOPO Cloning®-2 p. CR 8/GW/TOPO TA Cloning Kit $ price - One Shot TOP 10 # K 2500 -20 - One Shot Mach 1 -T 1 R # K 2520 -20 TOPO TA Cloning Kit + Gateway entry clone • Direct cloning with PCR product !! • Sequencing vector!! • Gateway Entry vector!! 7

Directional TOPO Cloning®-4 p. ENTR/TEV/D-TOPO Cloning Kit • With One Shot TOP 10 Chemically Competent E. coli Cat# K 2525 -20 • With One Shot Mach 1 -Tl Chemically Compentent E. coli Cat# K 2535 -20 TEV/Drectional TOPO Cloning with Gateway • Efficient cleavage of any N-terminal tag • No need to check orientation! • Gateway Entry vector!! 8

Vectors for Gateway® GOI 1 at t. L at t. P 2 2 Donor Vector att. B 2 t. L t. P GOI att. B 1 gene at at 1 ccd. B Entry Clone BP Reaction Kmr Km r LR Reaction Apr t. R 1 at 2 Expression Clone ccd. B t. R at 2 at gene t. B at t. B 1 GOI Destination Vector Apr 9

Modification of Recombination Creation of Direction Specific Selection att. B 1 att. B 2 x ccd. B att. P 1 BP reaction att. L 2 att. L 1 Kan. R x att. R 1 LR reaction att. R 2 Amp. R x att. R 2 Kan. R ccd. B att. L 2 att. L 1 att. P 2 ccd. B att. B 1 att. B 2 Amp. R x ccd. B att. P 1 att. P 2 Kan. R ccd. B: A gene encodes a protein that interferes with E. coli DNA gyrase. 10

Gateway PCR Cloning Highly efficient and HTP amenable with recombination method 11

Primer Design for att B PCR • Add the att B 1 sequence to the 5’-primer • Add the att B 2 sequence to the 3’-primer att B 1 Gene Specific Primer Sequence 5’ - GGGG ACA AGT TTG TAC AAA GCA GGC TNN NNN. . . att B 2 5’ - GGGG AC CAC TTT GTA CAA GAA AGC TGG GTN NNN. . . Gene Specific Primer Sequence 12

Invitrogen New Product Gateway BP Clonase II 1. Cat# and Price 이전제품 Gateway BP Clonase #11789 -013 (20 rxn) 427, 000원 #11789 -021 (100 rxn) 1, 807, 000원 최근 제품 Gateway BP Clonase II #11789 -020 (20 rxn) 185, 000원 #11789 -100 (100 rxn) 824, 000원 2. Contents BP Clonase Gateway BP Clonase Enzyme Mix 80 ul 5 X BP Clonase Reaction buffer 200 ul 2 ug/ul Proteinase K sol. 40 ul 30% PEG 8000/30 m. M Mgcl 2 sol. 1 ml 50 ng/ul Pexp 7 -tet Positive Control 20 ul BP Clonase II Gateway BP Clonase II Enzyme Mix 40 ul 2 ug/ul Proteinase K sol. 30% PEG 8000/30 m. M Mgcl 2 sol. 50 ng/ul Pexp 7 -tet Positive Control 40 ul 1 ml 20 ul 13

BP Protocol • att. B-PCR product (15~150 ng) • p. DONR vector • TE Buffer, p. H 8. 0 1 -7 l 1 l to 8 l 1. Add 2 l of BP Clonase™ II Enzyme Mix 2. Incubate for 1 hour at 25 o. C. 3. Add Proteinase K solution and incubate for 10 min at 37 o. C. 4. Transform competent E. coli 14

Cloning PCR Products a DNA mini-prep analysis p. UC = 108 CFU/ml 2 After overnight incubation 1 15

Gateway® Technology: Transfer Entry Clones into Expression Clones 16

Invitrogen New Product Gateway LR Clonase II 1. Cat# and Price 이전제품 Gateway LR Clonase #11791 -019 (20 rxn) 427, 000원 #11791 -043 (100 rxn) 1, 807, 000원 최근 제품 Gateway LR Clonase II #11791 -020 (20 rxn) 207, 000원 #11791 -100 (100 rxn) 874, 000원 2. Contents (20 rxn기준) LR Clonase Gateway LR Clonase Enzyme Mix 80 ul 5 X LR Clonase Reaction buffer 200 ul 2 ug/ul Proteinase K sol. 40 ul 50 ng/ul Pentr-gus Positive Control 20 ul LR Clonase II Gateway LR Clonase Enzyme Mix 40 ul 2 ug/ul Proteinase K sol. 40 ul 50 ng/ul Pentr-gus Positive Control 20 ul 17

LR Protocol • Entry Clone (50~150 ng) • Destination Vector (150 ng/ l) • TE Buffer, p. H 8. 0 1 -7 l 1 l to 8 l 1. Add 2 l of LR Clonase™ II Enzyme Mix 2. Incubate for 1 hour at 25 o. C. 3. Add Proteinase K solution and incubate for 10 min at 37 o. C. 4. Transform competent E. coli 18

Destination Expression Systems In vitro E. coli Mammalian Insect Native protein expression N-terminal fusion protein expression Yeast Viral C-terminal fusion protein expression Others 19

Parallel Transfer of CAT Gene into Multiple Destination Vectors Expression Vector Colonies Background Analysis Native (E. coli) 6 x. His-fusion GST-fusion Thioredoxin-fusion 15, 000 10, 650 9, 200 11, 000 0 0 4/4 4/4 Native protein (baculo) 6 x. His (baculo) 7, 800 6, 350 15 30 4/4 CMV-promoter Tet-regulated promoter 7, 950 6, 350 0 0 4/4 20

Gateway® reactions with Clonase™ II enzymes BP Clonase™ II reaction LR Clonase™ II reaction att. B-PCR product or linearized expression clone (~15 -150 ng) 1 -7 l Entry clone (~50 -150 ng) 1 -7 l Destination vector (150 ng) 1 l TE Buffer, p. H 8. 0 to 8 l p. DONR™ vector (150 ng) 1 l TE Buffer, p. H 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 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 21

Gateway® Systems Applications Drug discovery , Drug discovery assays 22

GATEWAY Collaborations for Building Source Clones • • • Harvard Institute of Proteomics (Harlow and La. Baer) – 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 23

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 24