Recombineering: versatile techniques to generate ... · Recombineering: versatile techniques to...

Recombineering: versatile techniques to generate recombinantDNA in pro- and eukaryotic cells

Michael HenselIRTG lecture 30.5.12

• direct manipulation of chromosomal genes• construction of reporter strains and chimeric genes• generation of scarless in frame deletions and insertions• manipulation of large DNA fragments (BAC)

Lecture overview

• background and motivation• principles of bacterial recombineering• application of bacterial recombineering

1. generation of mutant strains2. generation of bacterial reporters3. integration of expression cassettes4. marker-free manipulations5. recombineering of BAC for eukaryotic applications

• perspectives

Motivation

• approaches for directed genetic manipulation– restriction/ligation based gene cloning– application of PCR, splicing by overlap extension, etc.– gene synthesis, synthetic biology

• limitations– costs– maximal size of constructs– episomal vs. chromosomal genes– generation of constructs for applications in eukaryotic hosts

• e.g. targeting constructs for embryotic stem cells

• genetics by recombinogenic engineering - 'recombineering'• use of cell intrinsic mechanisms

– in yeast– in bacteria

Motivation - genetic engineering

• allelic exchange– conventional approaches– ineffcient in most bacteria

Gene X EcoRI cut sites

Kanamycin cassette

Cut with EcoRIand ligate

BamHIcut site

Cut with BamHIand transform intocell with wild-type

gene X

Linearized plasmid

Sites of recombination

Chromosome

Recombination and selectionfor kanamycin-resistant cells

Gene X knockout

Motivation

• giant, highly repetitive adhesins in bacterial pathogens– how to study a 595 kDa protein (5559 aa) with 53 domain repeats of Salmonella

enterica?

Background

• efficient recombination between linear DNA and chromosomal genes in yeast and few bacterial species - see IRTG lecture Jürgen Heinisch

• inefficient recombination between linear DNA and chromosomalDNA in E. coli and most other bacteria

– efficient degradation of linear DNA by exonucleases

• bacteriophages carry own recombination systems– efficient recombination of linear phages genomes into genome of host bacteria– only short homologous regions required– example - E. coli phage

• use of bacteriophage recombination systems to manipulate genes in bacteria?

– RecET– Red– ‚Red/ET cloning‘

Lecture overview



• perspectives

Principles of bacterial recombineering

• Reactions of recombination system ........Gam - prevents degradation of linear ds DNA by cellular RecBCD

lambdoid bacteriophages

genome of phage (integrated in E. coli genome)


• .... supplied by a bacterial expression system

• pKD46– ampicillin (carbenicillin) resistance

• bla– temperature sensitive ori

• oriR101, repA101ts– arabinose-inducible promoter

• PBAD

– repressor of PBAD

• araC– Red recombinase functions

• red , red exo– propagation in E. coli or others at ≤30°C with Carb., loss at ≥37°C

without Carb.


• .... supplied by modified genomes in E. coli strains

Lecture overview



• perspectives

Applications - deletions by recombineering

aph

aph

orfX

Red mediatedrecombination

PX

RBS

PX

RBS

transformation withlinear targeting DNA

orfX ::aph

PX

RBS orfX ::FRT

aphFor primer

Rev primer

PCR with 60 mer primers

FLP mediatedrecombination

Deletion ofSPI1 and SPI2 effector genesSPI1SPI2SPI3SPI4SPI9sadA locus......

in reference strains and clinicalisolates of -E. coliS. Typhimurium ATCC 14028S. EnteritidisS. Typhivarious Gram-negative species

aph

Datsenko and Wanner (2000) PNAS

scar sequence of 27 codons


• template plasmids for gene deletions (knockouts)– pKD3, pKD4 and pKD13– ampicillin (carbenicillin) resistance - bla– suicide plasmids, pir-dependent ori oriR

• replication only in pir-expressing strains– donor for resistance cassettes with

• kanamycin resistance - aph• chloramphenicol resistance - cat

– pKD13 for deletions avoiding polar effects


• design of oligonuceotides for mutagenesis– forward primer (Ec fliI Red13 Del for)– 5‘-CTGGCTAACCACGCTGGATAACTTTGAAGCCAAAATGGCGattccggggatccgtcgacc-3‘

– reverse primer (Ec fliI Red13 Del rev)– 5‘-CCAGCCCCTGGAGAGACGCTTCCCAGTCCGCGCGTTCAAAtgtaggctggagctgcttcg-3‘

– control primer forward– 5‘-cttaagtttgcatggctggc-3‘

– control primer reverse– 5‘-tgacatccgcgacgcatttc-3‘

complementary to target gene fliI (5‘) complementary to pKD13

complementary to target gene fliI (3‘) complementary to pKD13

Red recombineering - procedure

• Procedure I– electroporate pKD46 in target strain, maintain at 30°C with Carb

selection– grow strain [pKD46] in LB Carb at 30°C, add arabinose for

induction of Red functions• temperature is critical, use water bath shaker• purity of glassware and tubes is critical, use special

glassware and disposables– prepare competent cells

• growth phase is critical, harvest at OD600 of about 0.5 • purity of tubes is critical, use disposables• avoid change from 4°C during handling


• Procedure II– PCR amplify gene cassettes– purification (Qiagen)– DpnI digestion– purification (Qiagen or gel), quantification– electroporate linear DNA into competent pKD46 harboring target

strain– selection on LB at 37°C with kanamycin (pKD4, pKD13) or

chloramphenicol (pKD3)– clone purification, control of carbenicillin sensitivity– PCR confirmation of insertion by check PCR


• pCP20– Ampicillin (Carbenicillin) resistance

• bla– temperature sensitive ori

• oriR101, repA101ts– constitutive expression of FLP

recombinase– propagation in E. coli or Salmonella at ≤

30°C with Carb., loss at ≥ 37°C without Carb.


• Procedure III– prepare competent cells of clones of target strain with confirmed

gene knockouts (37°C)– electroporate with pCP20 – select on LB agar with carbenicillin et 30°C– streak purify clones on LB with carbenicillin and in parallel on LB

kanamycin (chloramphenicol), grow at 30°C– select kanamycin (chloramphenicol) sensitive clones – cure pCP20 by growth at 42°C (44°C)– confirm deletion of resistance cassette by check PCR

Applications - chromosomal epitope taggingby Red recombinase

aph

aph

orfX


PX

RBS


orfX::HA aph

aphFor primer

Rev primer



tagging with HA tagFLAG tag6 His tagetc. ......

aphPX

RBS

PX

RBSorfX::HA

Uzzau et al. (2001) PNAS

Applications - 'knock-in' of expressioncassettes

orfX


PX

RBS


aph

For primer

Rev primerPCR of expression cassettes


Pivi

antigen

aph

Pivi

aph

Pivi

Pivi

orfX ::aph

antigen

antigen

antigen

Husseiny & Hensel (2005) Infect. Immun.

• stable chromosomal integration of cassettes

• lack of resistance markers• simultaneous attenuation by

deletion• very rapid strain construction

0

1

2

3

4

5

6

Log 1

0C

FU/S

plee

n

Applications - gene fusions byrecombineering

reporter gene fusions

Red

FLP

reporteraph

aphreporter Z

orfX

reporter Z

orfX'

orfX'

PX

RBS

PX

RBS

PX

RBS

antigenaph

aph

orfX

Pivi

RBS

Pivi

RBS

PX

RBS

orfX'::antigen

orfX'::antigen

heterologous antigen fusions

Applications - reporter fusions byrecombineering

luc

GFPmut3

phoA

CAT

lacZ'

blaM

HaloTag©

p3121

p3131

p3126

p3142

p3138

p3143

p3141

FRT FRT common Rev primer

luc For primer

aph

SacISacII

NotIBamHI

SmaIEcoRI

EcoRVHindIII

ClaIHindII

NdeISalI

XhoI

MCSp2795

A B

Priming sites:

reporter-specific For primer:

luc type I: 5'- 40 nt target specific – ATGGAAGACGCCAAAAACATAA-3' luc type II: 5'- 40 nt target specific – GAAGACGCCAAAAACATAAGAA-3'

GFP type I: 5'- 40 nt target specific – ATGGCTAGCAAAGGAGAAGAAC-3' GFP type II: 5'- 40 nt target specific – GCTAGCAAAGGAGAAGAACTTT-3'

DsRed type I: 5'- 40 nt target specific –-ATGGCCACAACCATGGCCTCCTC-3' DsRed type II: 5'- 40 nt target specific –-GCCACAACCATGGCCTCCTCCG-3'

phoA type II: 5'- 40 nt target specific – CCTGTTCTGGAAAACCGGGC-3'

CAT type I: 5'- 40 nt target specific – ATGGAGAAAAAAATCACTGG-3'CAT type II 5'- 40 nt target specific – GAGAAAAAAATCACTGGATAT-3'

lacZ type II: 5'- 40 nt target specific – CCCGTCGTTTTACAACGTCG-3'

blaM type II: 5'- 40 nt target specific – CACCCAGAAACGCTGGTGAA-3'

HaloTag type II: 5'- 40 nt target specific –GGATCCGAAATCGGTACAGG-3'

common Rev primer:5'- 40 nt target specific – CGTGTAGGCTGGAGCTGCTTC-3'

DsRed p3176

Gerlach et al. (2007) Appl. Environ. Microbiol.

Applications - scarless/markerless deletionsby recombineering

orfX

1. Red-mediatedrecombination

PX

RBS

PX

RBS


orfX::tet

tetRFor primer

Rev primer


transformation with oligo2. Red-mediated recombination

tetA

tetR tetA

tetR tetA

PX

RBStetR tetA

selection of TetSclones

PX

RBS

i) Tet-based approach

80mer + X synthetic ds DNAanealed oligos introducing- aa exchanges- deletions- insertion- restriction sites- etc.

site-directed mutagenesis by recombineering

codon 498 499 500 501 502 503 504 505 506 507 508 509siiF WT gtg gta ggc gaa tgc gga gca gga aaa agc tca tta

V V G E C G A G K S S L

NlaIVsiiF G500E gtg gta gAA gaa tgc gga gcc gga aaa agc tca tta

V V E E C G A G K S S L

NlaIVsiiF K506L gtg gta ggc gaa tgc gga gcc gga TTa agc tca tta

V V G E C G A G L S S L

NlaIV SacIsiiF SacI gtg gta ggc gaa tgc gga gcc gga aaG agc tca tta

V V G E C G A G K S S L

A

B

C

WTNlaIV

G500EK506L

NlaIVNlaIV

siiF SacI

NlaIVNlaIV SacI

100 -

50 -

200 -

500 -

350 -

NlaIV SacI

M

• example – introduction of an aa exchange in the Walker A box of SiiF, the ATPase of the SPI4-T1SS

site-directed mutagenesis by recombineering

strain

WT siiE siiF # 1

# 1 [vector]

# 1 [comp.] # 2

# 2 [vector]

# 2 [comp.] # 4

# 4 [vector]

# 4 [comp.] # 1

# 1 [vector]

# 1 [comp.] # 3

# 3 [vector]

# 3 [comp.] # 2

# 2 [vector]

# 2 [comp.] # 3

# 3 [vector]

# 3 [comp.]

OD

450/

OD

600

0.00

0.05

0.10

0.15

0.20

siiF K506LsiiF G500E siiF SacI

• example – introduction of an aa exchange in the Walker A box of SiiF, the ATPase of the SPI4-T1SS

examples

N C

53 Ig domainrepeats

10 20 30 40 50

signalsequence

insertion

1

N-terminal moiety

2 3 4 5 6 71 8WT

2 3 4 5 6 71 8∆β-sheet

domain #2 MvP1249

Ig1

2 3 4 5 6 71∆coiled coilheptade 8 MvP1261

Ig1

2 3 4 5 61∆coiled coil

heptades 7-8 MvP1260

Ig1

2 3 41∆coiled coil


Ig1

5 6 7 8∆coiled coil


Ig1

3 4 5 6 7 8∆coiled coil


Ig1

∆coiled coilheptades 1-8

MvP1247Ig1

2 3 4 5 6 71 8∆1/3 β-sheet domain #1 MvP1258

Ig1

Ig1

2 3 4 5 6 71 8∆2/3 β-sheet domain #1 MvP1257

Ig1

WT

∆Ig 1-5 MvP1274∆Ig 1-4

MvP1273

∆Ig 1-2 MvP1271

∆Ig 1-3 MvP1272

∆Ig 1 MvP1270

D

20 41

∆Ig 21-25 MvP1227∆Ig 21-30 MvP1226

∆Ig 21-40 MvP1222

5347

WT

∆Ig 48-52 MvP1269∆Ig 49-52 MvP1268∆Ig 50-52 MvP1267∆Ig 51-52 MvP1266

∆insertionMvP1400

∆1/2Ig 52 MvP1252

∆Ig 53 MvP1255

∆ins-Ig53 MvP1399

∆Ig 52 MvP1251

∆1/2Ig 53 MvP1254∆Ig 53 1-52 MvP1265∆Ig 53 1-42 MvP1264

∆Ig 53 1-29 MvP1263∆Ig 53 1-15 MvP1262

Wagner et al. (2011) Cell. Microbiol.

examples

• insertion by Red-recombineering

N-terminale Sequenz

β-Faltblatt-domäne #1

β-Faltblatt-domäne #2

coiled coilDomäne

Ig-Domäne 1

Ig-Domäne 2

LKKQLDD

AENAKKE

ADKAKEE

AEKAKEA

AEKALNE

AFEVQNS

SKQIEEM

LQNFLAD

A B

LKKQLDD

AENAKKE

ADKAKEE

AEKAKEA

AEKALNE

AFEVQNS

SKQIEEM

LQNFLAD

AEKALNE

AFEVQNS

SKQIEEM

LQNFLAD

Heptade 1Heptade 2Heptade 3Heptade 4Heptade 5Heptade 6Heptade7Heptade 8

Heptade 5Heptade 6Heptade 7Heptade 8

C

Polke (2010) Diploma thesis

Red-mediated scarless in-framemutagenesis – tetAR replacement

orfX

C site-directed mutagenesis or insertion

RedPX

RBS

tetA tetR

orfX'

PX

RBS tetA tetR

PX

RBStetA tetR

orfX'

orfX mut

PX

RBS

Red

PX

RBStetA tetR

PX

RBS

Red

orfX'orfX' orfX'orfX'

B in-frame deletion

A insertion of tetAR cassette

orfX mut

Check-Rev Check-FororfX-For orfX-Rev

Gerlach et al. (2009) Appl. Environ. Microbiol.

scarless deletions by Red recombinase

orfX

1. Red-mediatedrecombination

PX

RBS

PX

RBS

orfX::I-SceI aph

transformation with oligo2. Red-mediated recombination

PX

RBS

expression of I-SceIselection of survivors

PX

RBS

ii) I-SceI-based approach

aph transformation withlinear targeting DNA

aphFor primer

Rev primer


I-SceI

aph

aph

I-SceI recognition site -cut by I-SceI andgeneration of double strandbreaks in chromosome

Red-recombineering with I-SceI

• I-SceI– intron encoded ‚homing‘ endonuclease from

Saccharomyces cerevisiae– cloned and expressed in E. coli– rare recognition sequence of 18 bp – mega-nuclease

Meganuclease-induced double strand breaks(DSB)

• DSB– stop of DNA replication, lethal to cells– various repair mechanisms

Approach for I-SceI-based recombineering

Blank et al. (2011) PLoS One

proof of principle

– mutagenesis of Salmonella phoQ– central virulence regulator, PhoPQ two-component system– reconstruction of pho-24, a.k.a. phoPc, a.k.a phoQ T48I

Lecture overview



• perspectives

Application - modification of BAC

• bacterial artificial chromosomes (BAC)– bacterial vectors for cloning and manipulation of large DNA

fragments up to 300 kb in E. coli• for generation of constructs for ES knock-out in mice

• for generation of ES reporter constructs

• introduction of point mutations

commercial provider:

Application - modification of C. elegans

• bacterial artificial chromosomes (BAC)

Lecture overview



• perspectives

Perspectives

• Recombineering in bacteria:– broad application to manipulation of genes of bacterial or

eukaroytic origin– PCR/DNA synthesis based generation of constructs– only basic handling of E. coli required

• transformation• DNA extraction

– versatile technique for generation of knock-outs, fusions, chimeric genes, site directed mutants, etc.

– cheap and rapid

Literature

• Initial descriptions– Zhang, Y., Buchholz, F., Muyrers, J.P., and Stewart, A.F. (1998). A new logic for DNA engineering using

recombination in Escherichia coli. Nat Genet 20, 123-128.– Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-

12 using PCR products. Proc Natl Acad Sci U S A 97, 6640-6645.– Yu, D., Ellis, H.M., Lee, E.C., Jenkins, N.A., Copeland, N.G., and Court, D.L. (2000). An efficient

recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97, 5978-5983.

• Modifications– Husseiny, M.I., and Hensel, M. (2005). Rapid method for the construction of Salmonella enterica Serovar

Typhimurium vaccine carrier strains. Infect Immun 73, 1598-1605.– Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M. (2007). Rapid engineering of bacterial reporter gene

fusions by using Red recombination. Appl Environ Microbiol 73, 4234-4242.– Gerlach, R.G., Jäckel, D., Holzer, S.U., and Hensel, M. (2009). Rapid oligonucleotide-based recombineering

of the chromosome of Salmonella enterica. Appl Environ Microbiol 75, 1575-1580.– Blank, K., Hensel, M., and Gerlach, R.G. (2011). Rapid and highly efficient method for scarless mutagenesis

within the Salmonella enterica chromosome. PLoS ONE 6, e15763.

Literature

• Protocols– Sharan, S.K., Thomason, L.C., Kuznetsov, S.G., and Court, D.L. (2009). Recombineering: a homologous

recombination-based method of genetic engineering. Nature protocols 4, 206-223.– Sarov, M., Schneider, S., Pozniakovski, A., Roguev, A., Ernst, S., Zhang, Y., Hyman, A.A., and Stewart, A.F.

(2006). A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature methods 3, 839-844.

– Bird, A.W., Erler, A., Fu, J., Heriche, J.K., Maresca, M., Zhang, Y., Hyman, A.A., and Stewart, A.F. (2012). High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificialchromosomes. Nature methods 9, 103-109.

– Methods Enzymol. Vol. 477

• Commercial supplier (example)– http://www.genebridges.com/

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