Transcript Die Achterbahn - Swansea University

Lambda RED Recombination
JIC in Norwich
11.07.2005
University of Tübingen
Bertolt Gust
Genomic organisation of the lambdoid bacteriophage
Genes exo, bet and gam are clustered in the
PL operon.
red- (recombination defective)  mutants
were partially defective in homologous
recombination in a wt-host, and grossly
defective in a recA- host.
The E. coli recombination system primarily
restores collapsed replication forks, repairs
DSB and maintains the genetic integrity of
the E. coli chromosome.
During the replication of , the infected cell
is a hotbed of genetic exchange (hyper-rec
state).
11.07.2005
Bertolt Gust
How does  Red stimulate homologous recombination ?
linear DNA
5´
3´
3´
5´
5´
3´
3´
5´
circular DNA
11.07.2005
Bertolt Gust
 Gam () inhibits the exonuclease V activity of the
recBCD system
Rec
BCD
linear DNA


5´
3´
3´
5´


5´
3´
3´
5´
Rec
BCD
circular DNA
 Gam () binds as a dimer to the E. coli RecBCD complex and inhibits its nuclease activity
11.07.2005
Bertolt Gust
 Exo () binds to dsDNA ends ...

linear DNA

3´
5´
5´
3´
Subramanian et al., 2003
5´
3´
3´
5´
circular DNA
  Exo () degrades linear dsDNA in 5´ to 3´direction (1kb/sec in-vitro) leaving long
3´ssDNA overhangs
 The active form of the protein (24kDa) is a trimer with a central hole
11.07.2005
Bertolt Gust
... and progressively generates 3´ overhangs

linear DNA
3´

3´
Subramanian et al., 2003
5´
3´
3´
5´
circular DNA
The entrance of the hole accommodates dsDNA, the exit diameter is the size of ssDNA
11.07.2005
Bertolt Gust
 Beta (ß) binds to ssDNA and mediates invasion of the
ssDNA into an unbroken homologous duplex
linear DNA
3´
ß ß ß ß
ß ß ß ß
3´
5´
3´
3´
5´
circular DNA
  Beta (ß) binds to ssDNA greater than 35 nucleotides in length
 Beta belongs to a family of recombination proteins which include Erf protein of
Salmonella phage P22, the RecT protein of the cryptic E. coli phage Rac, and the Rad52
protein of eukaryotes
 Beta promotes denaturation of complementary strands, strand annealing and exchange
reactions
11.07.2005
Bertolt Gust
RecFOR is essential for the formation of the
recombination complex
linear DNA
RecFOR
5´
3´
3´
ß ß ß ß
ß ß ß ß
3´
3´
5´
circular DNA
RecFOR
 RecFOR replaces single-strand binding proteins (SSB) bound on ssDNA with RecA
 RecA stabilises complex of Bet, DNA and RecFOR
11.07.2005
Bertolt Gust
RuvAB helicase-driven branch migration results in
Holliday junction formation ...
linear DNA
Rafferty et al., 1996
RuvAB
5´
3´
3´
ß ß ß ß
ß ß ß ß
3´
3´
5´
circular DNA
RuvAB
RuvAB recognizes a four-way junction ( Holliday junction) and catalyzes branch migration
11.07.2005
Bertolt Gust
... which can be resolved by RuvC ...
linear DNA
RuvC
RuvC
Rafferty et al., 1996
5´
3´
3´
ß ß ß ß
ß ß ß ß
3´
3´
5´
circular DNA
RuvC is a Holliday junction endonuclease (structure specific resolvase)
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Bertolt Gust
... into a recombinant molecule
11.07.2005
5´
3´
3´
5´
Bertolt Gust
PCR-targeting (step 1)
oriT marker
FRT
X
P1
FRT
39bp
P2
target
39bp
X
PCR product
S. coelicolor cosmid
neo
oriT marker
FRT
FRT
P1
SuperCos1
bla
P2
S. coelicolor cosmid
neo
11.07.2005
bla
Bertolt Gust
PCR-targeting (step 2)
target
oriT marker
FRT
P1
FRT
X
P2
X
S. coelicolor
chromosome
S. coelicolor cosmid
neo
SuperCos1
oriT marker
FRT
11.07.2005
FRT
P1
bla
P2
S. coelicolor
chromosome
Bertolt Gust
Why two-step strategy?
1.  Red is efficient in E. coli
2. Recombinant cosmid-DNA can easily be confirmed by PCR,
restriction analysis and/or sequencing
3. Mutagenised cosmids can be mobilised by conjugation, no
need for transformation procedures
4. High frequency of double cross-overs due to long flanking
sequences in a cosmid clone
Disadvantages
1. Dependent upon the availability of a E. coli clone
2. Not high throughput (in comparison to transposon
mutagenesis)
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Bertolt Gust
Template cassettes for gene replacements
pIJ782
P1
FRT
pIJ797
P1
FRT
SwaI
P1 oriT aac(3)IV P2
pIJ776
P1
FRT
pIJ777
P1
FRT
pIJ778
P1
FRT
pIJ779
P1
FRT
neo
neo
P2
FRT
FRT
aadA
oriT
oriT
P2
tet
hyg
P2
P2
NEW
P2
oriT aadA
FRT
11.07.2005
oriT
FRT
pIJ775
vph
vph
FRT
P1
P2
FRT
pIJ781
vph
FRT
P2
oriT
FRT
P1
FRT
SwaI
pIJ780
FRT
oriT aac(3)IV
P2
loxP
P1
FRT
pIJ774
oriT aac(3)IV
loxP
P1
FRT
pIJ773
P2
P2
Bertolt Gust
Template cassettes for other applications
pMS80
P1
oriT aac(3)IV
P2 tcp
oriT aac(3)IV
FRT
P1
P2 fd-ter nitAp
FRT
egfp
bla
P2 tipAp
FRT
pIJ786
P1
aac(3)IV oriT
FRT
NEW
aac(3)IV oriT
FRT
P1
int
attP
FRT
pIJ785
tet
FRT
bla oriT
FRT
pIJ787
P2
pIJ784
bla oriT aac(3)IV bla
pIJ798
bla oriT
pIJ789
neoaac(3)IV neo
pIJ794
neo oriT aac(3)IV neo
pIJ795
neo oriT
pIJ796
neo oriT aadA
bla
hyg
NEW
neo
vph
neo
NEW
S. coelicolor cosmid
neo
bla
Herai et al., 2004. Hyper-inducible expression system for streptomycetes. PNAS 101, 14031-14035
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Bertolt Gust
REDIRECT (Rapid Efficient Directed Recombination Time saving)
Download protocol and Primer design program at http://streptomyces.org.uk/redirect/index.html
To obtain the REDIRECT KIT: Mail Nicholas Bird [email protected]
USA
United Kingdom
Germany
Canada
Spain
China
Korea
Netherlands
Japan
Taiwan
Argentina
Belgium
Finland
France
Israel
Sweden
Switzerland
Taiwan
26
21
12
7
7
5
4
2
2
2
1
1
1
1
1
1
1
1
Gust, B., Chandra, G., Jakimowicz, D., Tian, Y., Bruton, C.J. and Chater, K.F. (2004) λ Red-mediated genetic manipulation of antibiotic-producing
Streptomyces, Advances in Applied Microbiology 54: 107-28.
Gust, B. Challis, G.L., Fowler, K., Kieser, T. and Chater, K.F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain
needed for biosynthesis of the sesquiterpene soil odour geosmin, Proc. Natl. Acad. Sci. USA 100: 1541-6
Gust, B., Kieser, T. and Chater, K.F. (2002) REDIRECT technology: PCR targeting system in Streptomyces coelicolor, John Innes Centre,
Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom
11.07.2005
Bertolt Gust
Epitope tagging using REDIRECT
a
b
aac(3)IV oriT
FRT
FRT
P1
1. PCR ab
P2
c
aac(3)IV oriT
FRT
P1
FRT
Tag
P2
2. PCR ac
cosmid
neo
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bla
Bertolt Gust
Epitope tagging using REDIRECT
a
b
aac(3)IV oriT
FRT
FRT
P1
1. PCR ab
P2
c
aac(3)IV oriT
FRT
P1
FRT
Tag
P2
2. PCR ac
cosmid
neo
11.07.2005
bla
Bertolt Gust
Introducing point mutations using REDIRECT
pIJ775
P1 aac(3)IV oriT P2
SwaI
40 bp
40 bp
*
X
I-SceI SwaI
*
PCR-product
P1
P2
X
cosmid
neo
bla
select for KanR CarbR transformants
11.07.2005
Bertolt Gust
Single strand oligonucleotide repair (ssOR)
Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells
Youming Zhang, Josep PP Muyrers, Jeanette Rientjies and A. Francis Steward
BMC Molecular Biology 2003, 4:1-14
• Only λ Bet is required
• Strand bias: more ssOR with oligos priming the lagging strand
• Efficiency of ssOR is maximum with oligos ~ 120 nt
leading
5´
3´
DnaB
5´
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DNA Pol III
5´
3´
5´
lagging
Bertolt Gust
Oligo-Targeting for deleting transposon insertions
120bp dsDNA
Afl II
Tn5062
S. coelicolor cosmid
neo
bla
S. coelicolor cosmid
neo
11.07.2005
bla
Bertolt Gust
Oligo-Targeting for generation of “scar less” in-frame
deletions
120bp dsDNA
I-SceI
Cyc2 P1 oriT aac(3)IV P2
S. coelicolor cosmid
neo
bla
S. coelicolor cosmid
neo
11.07.2005
bla
Bertolt Gust
Summary
PCR-targeting
gene knock-outs
gene replacements
gene fusions
module swapping
epitope tagging
inserting point
mutations
promoter replacements
ET-cloning
integration of cosmids
(attP)
promoter replacements
11.07.2005
Oligo-targeting
generating point mutations
inserting restriction sites
“scarless” mutations
N-terminal epitope tagging
deletion of transposon insertion
Bertolt Gust
Acknowledgements
Department of Molecular Microbiology
Celia Bruton
Prof. Keith Chater
11.07.2005
Prof. Mervyn Bibb
Sir David Hopwood
Mark Buttner
Tobias Kieser
Greg Challis
Helen Kieser
Kay Fowler
Prof. Barry
Bertolt
Wanner
Gust