Gateway

Technology

R-DNA Technology
M.Phil. Biotechnology
1st Semester
CBM, University of Swat
 Table of Contents
S. No. Title P. No.
1 Introduction 3
2 How it works? 4
3 Phage λ Recombination System in E. coli 5
4 Gateway Cloning 9
5 Integrase’s Function
6 BP Reaction 10
7 LR Reaction 11
8 Advantages 12
9 Applications 13
 Introduction

– Invented and commercialized by Invitrogen life technologies since the late 1990s
– Efficiently transfer DNA fragments between plasmids using an appropriate set of
recombination sequences, the "Gateway att" sites, and two proprietary enzyme mixes,
called "LR Clonase", and "BP Clonase"
– Based on site specific recombination system of phage λ
– Directional cloning
– Maintains reading frame
– No restriction & No ligation enzymes
– 1 hour, room-temperature reaction with >99% efficiency
– No re-sequencing
– Compatible with automation
– Reversible reactions
How it works?

DNA
Material

Add mixture
Plate the
to
Incubate for reaction &
competent
Buffer 1hr at room incubate
cells & allow
temperature overnight at
for 45min to
37°C
transform

Clonase
enzyme
Phage λ Recombination System in E. coli
cos

Phage l
attP ꟷ Insertion of phage DNA into E.coli
232 bp
genomic DNA

+ ꟷ attP site of phage and attB site of

attB E.coli
E. coli
21 bp
ꟷ 2 molecules i.e. integrase (Int) &
integration host factor (IHF)
Integration Excision
(Int, IHF) (Int, IHF, Xis)

attL attR
Lysogen
96 bp 157 bp
Phage λ Recombination System
in E.coli
attB ACAAGTTTGTACAAAAAAGCAGGCT
TGTTCAAACATGTTTTTTCGTCCGA
+
attP N75CCAACTTTGTACAAAAAAGCTGAACN100
N75GGTTGAAACATGTTTTTTCGACTTGN100
BP Clonase (Int + IHF) LR Clonase (Int + IHF + Xis)

attL N75CCAACTTTGTACAAAAAAGCAGGCT
N75GGTTGAAACATGTTTTTTCGTCCGA
+
attR ACAAGTTTGTACAAAAAAGCTGAACN100
TGTTCAAACATGTTT TTTCGACTTGN100
 Integrase’s Function

attP
Integarse dimers binds to att sites

attB

attP
Integrase tetramerizes to form synaptic complex
attB
 Integrase’s Function

attP attP attL

Rotation
Cleavage & Ligation

attB attB attR

Protein-DNA complex Strand swapping Ligation & Release
formation
Gateway Cloning

Includes two main reactions

i.e. BP reaction and LR reaction
 BP Reaction

Primer
GOI ccdB GOI
attP1 attP2 attL1 attL2
PCR Primer
Donor attR1 ccdB attR2 Entry
vector clone
attB1 attB1 By-product
Kan Kan

attB – PCR product + Donor vector + BP clonase Transformation

(1hr Incubation)

Screening

>90 – 99%
correct clones
LR Reaction

CaMPARI
attL1 attL2 CaMPARI
attB1 attB2
pENTR4 Entry clone
Expression
vector
Kan LR Clonase
Amp
ccdB
attR1 attR2 ccdB
attP1 attP2
pDEST15 Donor
Destination clone vector
Amp
Kan
Advantages

– Efficiently and easily shuttle insert DNA from one plasmid to another
– Simplify the cloning workflow and save time
– Create expression clones without using restriction and ligase enzymes
– Simultaneously clone, in a specific order and orientation, up to 4 DNA
fragments into one plasmid
 Applications

Your
– Gene Acquisition PCR
Source
Library
– Drug target identification
ORF Gene
– Cloning collection synthesis
– Sequencing
Gene
– Cloning & sub-cloning Gene Gene
– Building clone & library collections Protein Your Application
– Delivery Localization

– Transfection into challenging mammalian cell lines Gene

– In vivo studies in animal models Gene Entry Clone Gene

– Protein Production Protein

Purification RNAi
– Protein arrays
– Antibody or antigen production Gene Gene
– Protein Analysis Cell-Free Protein
– Interactions interaction

– Reporter Assays
– Localization
– RNAi Gene1 Gene2 Gene3 Gene4
– Purification Your Application
– Functions