Gateway® Technology:
A Conduit to Proteomics
KDR Biotech Co., Ltd.
The Cloning Bottleneck
Issues
1) Restrictions Sites
5'
GOI
3'
2) Multiple steps/enzymes
PCR
3) Time
4) Compatibility
Cut & Ligation
Cloning Vector
5) Not HTP
GOI
Cut & Ligation
Cut & Ligation
Exp Vector 1
Cut & Ligation
Exp Vector 2
Exp Vector N
2
Designing a New Approach to Cloning
Gene
•
•
•
•
•
Maximize compatibility and flexibility
Minimize planning
Maintain reading frame
Rapid
– No restriction enzymes
– No gel purification
– No ligations
High-throughput
Gene
3
Subcloning an Entry Clone into Multiple Destinati
on Vectors
Gene
Gene
In vitro translation
Fusion protein
Gene
Viral
Gene
Gene
Gene
E. coli
Entry Clone
Your Vector
Gene
Gene
Yeast 2-Hybrid
Gene
Mammalian
Insect
4
Building Entry Clones
Three Options
Conventional cloning
Directional
TOPO® Cloning
Gateway®
PCR Cloning
Vector
MCS
gene
Entry
Vectors
L1
ccdB
L2
TOPO®-activated
Entry Vector
Donor
Vector
Kmr
Kmr
Kmr
Insert
Restriction
fragment
B1
PCR Product
B2
attB PCR
Product
5
Directional TOPO Cloning®-1
Fast and simple directional PCR cloning strategy with >90% efficiency
6
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!!
7
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!!
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Vectors for Gateway®
ccdB
GOI
gene
attB1
Donor
Vector
GOI
attB2
Entry
Clone
BP Reaction
Kmr
Km r
GOI
gene
Expression
Clone
Apr
LR Reaction
ccdB
Destination
Vector
Apr
9
Modification of Recombination
Creation of Direction
Specific Selection
BP reaction
ccdB
attB1
attB2
x
attP1
ccdB
attL2
attL1
KanR
x
attR1
attR1
LR reaction
attR2
AmpR
x
attR2
KanR
KanR
ccdB
attL2
attL1
attP2
ccdB
attB1
attB2
AmpR
x
attP1
attP2
KanR
ccdB: A gene encodes a protein that interferes with E. coli DNA gyrase.
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Gateway PCR Cloning
attB1
attB2
ccdB
Gene
+
2
tP
at
at
tP
1
Gene
Donor
Vector
PCR Product
Kmr
gene
2
Entry
Clone
> 90-99% correct
clones
tL
at
at
tL
1
BP Reaction
+
ccdB
By-Product
Kmr
Highly efficient and HTP amenable with recombination method
11
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
Gene Specific
Primer Sequence
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
12
Invitrogen New Product
Gateway BP Clonase II
1. Cat# and Price
이전제품
Gateway BP Clonase
#11789-013 (20rxn) 427,000원
#11789-021 (100rxn) 1,807,000원
최근 제품
Gateway BP Clonase II
#11789-020 (20rxn) 185,000원
#11789-100 (100rxn) 824,000원
2. Contents
BP Clonase
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
BP Clonase II
Gateway BP Clonase II Enzyme Mix 40ul
2ug/ul Proteinase K sol.
30% PEG 8000/30mM Mgcl2 sol.
50ng/ul Pexp7-tet Positive Control
40ul
1ml
20ul
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BP Protocol
• attB-PCR product (15~150ng)
• pDONR vector
• TE Buffer, pH 8.0
1-7 ml
1 ml
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
Size (kb) PCR DNA PCR DNA
(fmol)
(ng)
Colonies/ml
Transformation1
15
38
15
38
15
38
15
38
15
38
15
38
7.5
37.5
1223
2815
507
1447
271
683
478
976
190
195
30 (2352)
54 (4632)
16 (1122)
42 (2012)
0.26
1.0
1.4
3.4
4.6
6.9
10.1
a
3
7.5
10
25
14
35
34
85
46
115
69
173
50.5
252.5
Correct
Clones/Total
Clones Examined
10/10a
49/50
48/50
9/10a
10/10a
47/50
15/16
DNA mini-prep analysis
1
pUC = 108 CFU/ml
2 After overnight incubation
15
Gateway® Technology: Transfer Entry Clones into
Expression Clones
Entry
Clone
+
Kmr
2
tR
at
2
at
tR
1
gene
tL
at
at
tL
1
ccdB
Destination
Vector
Apr
LR Reaction
LR Clonase™ Mix
Apr
1
at
tP
+
2
Expression
Clone
tP
at
gene
2
tB
at
at
tB
1
ccdB
By
Product
Kmr
16
Invitrogen New Product
Gateway LR Clonase II
1. Cat# and Price
이전제품
Gateway LR Clonase
#11791-019 (20rxn) 427,000원
#11791-043 (100rxn) 1,807,000원
최근 제품
Gateway LR Clonase II
#11791-020 (20rxn) 207,000원
#11791-100 (100rxn) 874,000원
2. Contents (20rxn기준)
LR Clonase
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
LR Clonase II
Gateway LR Clonase Enzyme Mix 40ul
2ug/ul Proteinase K sol.
40ul
50ng/ul Pentr-gus Positive Control 20ul
17
LR Protocol
• Entry Clone (50~150ng)
• Destination Vector (150ng/ml)
• TE Buffer, pH 8.0
1-7 ml
1 ml
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
18
Destination Expression Systems
In vitro
E. coli
Native protein expression
Mammalian
Insect
N-terminal fusion protein expression
Yeast
C-terminal fusion protein expression
Viral
Others
19
Parallel Transfer of CAT Gene into Multiple Destination Vectors
Expression Vector
Colonies
Background
Analysis
Native (E. coli)
6xHis-fusion
GST-fusion
Thioredoxin-fusion
15,000
10,650
9,200
11,000
0
0
0
0
4/4
4/4
4/4
4/4
Native protein (baculo)
6xHis (baculo)
7,800
6,350
15
30
4/4
4/4
CMV-promoter
Tet-regulated promoter
7,950
6,350
0
0
4/4
4/4
20
Gateway® reactions with Clonase™ II enzymes
BP Clonase™ II reaction
LR Clonase™ II reaction
attB-PCR product or linearized expression
clone (~15-150 ng)
1-7 ml
Entry clone (~50-150 ng)
1-7 ml
Destination vector (150 ng)
1 ml
TE Buffer, pH 8.0
to 8 ml
pDONR™ vector (150 ng)
1 ml
TE Buffer, pH 8.0
to 8 ml
Vortex BP Clonase™ II and add 2 ml to above
Incubate at 25C for 1 hour
Incubate in 1 ml Proteinase K at 37 C for 10 min
Vortex LR Clonase™ II and add 2 ml to above
Incubate at 25C for 1 hour
Incubate in 1 ml Proteinase K at 37 C for 10 min
Transform
Transform
Clonase™ II reactions are 10 ml volumes instead of 20 ml
21
Gateway® Systems Applications
Table 1Gateway® Vectors and Systems
Stage of Research
Gene Acquisition
Cloning
Delivery
Protein Production
Protein Analysis
Drug discovery
Application
Drug target identification
Sequencing
Cloning & subcloning
Building clone & library collections
Transfection into challenging mammalian
cell lines
In vivo studies in animal models
Protein arrays
Antibody or antigen production
Function
Interactions
Reporter Assays, Drug discovery assays
Localization
RNAi
Purification
Gateway® Products
Ultimate™ ORF Clone collection
Entry and donor vectors
CloneMiner™ cDNA Library Construction Kit
ViraPower™ Expression Systems
ViraPower™ Lentiviral Expression Systems
Expressway™ Plus Expression System
Champion™ pET vectors
BaculoDirect™ Expression System
pcDNA™ mammalian vectors
Two-hybrid systems
GeneBlazer™
Lumio Technology and GFP
Block-It™ RNAi Technology
Tag-on-Demand™ Technology
22
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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