In vivo recombination and yeast genetics as tools · 2017-02-28 · AG Genetik IRTG-Seminar - April...

IRTG-Seminar - April 25, 2012 Jürgen J. HeinischAG Genetik

In vivo recombination and yeast genetics as tools

for studying microcompartmentation

IRTG-Lecture

Jürgen J. HeinischApril 25, 2012


Lecture overview

Shuttle vectors and use of yeast for in vivo cloning

Multiple deletions and tetrad analysis for combination of desired traits

Substitution of deletion cassettes for in vivo mutated genes

“Beyond the rim“: C- and N-terminal tags, yeast display, gap-repair, and many more


Characteristics of shuttle vectors (2-way/double)

- maintenance and expression in YFO (= “Your Favorite Organism“)- suitable cloning sites- selection and amplification in E. coli

YEp352

5407 bps

1000

2000

3000

4000

5000

Eco RI 230Sma I 246BamHI 251Xba I 257Sal I 263Sph I 275

Bcl I 847Nhe I 891

Nde I 1206

I 3910Sca

orilacZα

URA3

2µm

bla

yeast replication

yeast selection

E. coliselection

E. colireplication

multiplecloning

site

L22_eGFP9987 bps

2000

40006000

8000

Spe I 252Nde I 486

Bam HIEco RI 4655I 5283Xho

I 9545Sca

CMV

LTR5'Psi

CamKII

eGFPWPRELTR3'

f1 ori

SV40 ori

oribla

E. colireplication

E. coliselection

mammalian propagation

expressionsignals

YFG(= Your Favorite Gene)

cloningsite

Yeast/E. coli Lentivirus/E. coli


Frequent problems with available shuttle vectors- limited number of suitable cloning sites (e.g. only BamHI)

- problems with classical cloning by restriction/ligation- restriction site may also appear within the gene of interest to

be expressed

- restriction site composition may interfere with translation efficiency of mRNA generated from a cDNA clone

BamHI BglII BclI

XmaI

YFG-ORF

------ C C C G G G AUG ------

Met – Aa – Aa – Aa ------

mRNA

Protein


The solution: Cloning by in vivo recombination (IVR)

L22_eGFP9987 bps

2000

40006000

8000

Spe I 252NdeI 486

Bam HIEcoRI 4655I 5283Xho

I 9545Sca

CMV

LTR5'Psi

CamKII

eGFPWPRELTR3'

f1 ori

SV40 ori

oribla

target sequence linearized with BamHI

YFG

YFG

PCR product

gene cloned into expression vector

CamKII promoter

CamKII promoter

terminator

terminator

The principle:

Yeast, Saccharomyces cerevisiae, is the best tool for IVR cloning(fast, efficient, accurate)


A step-by-step procedure for the construction and use of triple shuttle vectors

1. IVR cloning requires a replication origin and a selection marker for yeast

YEp3525407 bps

1000

2000

3000

4000

5000

EcoRI 230SmaI 246BamHI 251XbaI 257Sal I 263SphI 275

BclI 847NheI 891

NdeI 1206

I 3910Sca

orilacZα

URA3

2µm

bla

L22_eGFP9987 bps

2000

40006000

8000

Spe I 252NdeI 486

Bam HIEcoRI 4655I 5283Xho

I 9545Sca

CMV

LTR5'Psi

CamKII

eGFPWPRELTR3'

f1 ori

SV40 ori

oribla

URA3300 bp 40 bp2µm

bla 5‘

Oligo 1

Oligo 2

pJJH1242

12609 bps

2000

4000

6000

8000

10000

12000

Nde I 1165

Spe I 2257

Nde I 2491

Not I 3529

Bam HI 5899I 6645Not

RI 6660Eco

I 7288Xho

URA3

CMV

CamKII

eGFPWPRE

f1 ori

SV40 ori

pUC ori

bla

2µm



1b. Yeast sequences for Drosophila vectors

pUAST9050 bps

2000

4000

6000

8000

Sph I 1Eco RI 380Bgl II 392Not I 398

BamHI 1278

Eco RV 4279

I 5317Nsi

I 8264Nde

HI 9039Bam

UAS-HSPp

white-SV40

bla

pJJH13965122 bps

1000

20003000

4000

5000

PstI 35Sal I 41

XbaI 95

Stu I 571

NcoI 800EcoRV 819

RV 2712Eco

URA3

2µmloxP

bla

loxP

Oligo 1

Oligo 2

URA3 40 bp40 bp 2µm

loxPloxP

pJJH141011762 bps

2000

4000

6000

8000

10000

Sph I 1Eco RI 380Bgl II 392Not I 398

BamHI 1278

Eco RV 4279

Nsi I 5317

I 8423Nsi

RV 9060Eco

I 9990Nsi

RV 10953EcoHI 11751Bam

UAS-HSPp

white-SV40

bla

loxP

URA3

2µmloxP



1c. Yeast sequences for insect vectors

pJJH14608637 bps

2000

4000

6000

8000

BamHI 1Eco RI 23Sal I 39Not I 58Pst I 81

Pst I 3440Sal I 3446

Eco RV 4224

RV 6117EcoRV 6245Eco

I 7596KpnI 7602Sph

I 8343Xho

bla

ori

loxP

URA32µm

loxP

Gentamycin

P10/PHp

pFastBac dual5238 bps

1000

20003000

4000

5000

RV 2923Eco

I 4280SphI 4287NsiI 4298NheI 4311Xho

HI 4606BamRI 4628Eco

I 4644SalI 4656SpeI 4663NotI 4678Xba

bla

oriGentamycin

P10PHp

SV40PolyA



2a. IVR cloning of genes of interest coupled to a selectable marker

pJJH124212609 bps

Nde I 1165

Spe I 2257Nde I 2491

Not I 3529


RI 6660EcoI 7288Xho

URA3

CMV

CamKII

eGFPWPRE

f1 ori

SV40 ori

pUC ori

bla

2µm

target vector linearized with BamHI

YFG

YFG

PCR product fromplasmid template withgene coupled to marker


CamKII promoter

CamKII promoter

terminator

terminator

markere.g. kanMX (Geneticin)

KlLEU2SkHIS3

marker

Co-transformation of linearized (optional) vector (0.1-2 µg) with PCR product (5-10 µg) into S. cerevisiae

Selection for marker(e.g. on plates with Geneticin or

lacking leucine/histidine)

Oligonucleotides for PCR reaction:

YFG

marker

40 nt 20 nt

20 nt 40 nt

target vector (3‘)

target vector (5‘)5‘

3‘

3‘

5‘



2b. IVR cloning of genes of interest directly from cDNA

pJJH124212609 bps

Nde I 1165


Not I 3529


RI 6660EcoI 7288Xho

URA3

CMV

CamKII

eGFPWPRE

f1 ori

SV40 ori

pUC ori

bla

2µm


YFG

YFG

PCR product fromplasmid template withgene coupled to marker


CamKII promoter

CamKII promoter

terminator

terminator

Co-transformation of linearized (mandatory) vector (10-50 ng) with PCR product (5-10 µg) into S. cerevisiae

Selection for vector (on plates lacking uracil)


YFG

YFG

40 nt 20 nt

20 nt 40 nt



3‘

3‘

5‘



2c. IVR cloning of genes of interest with C-terminal eGFP-tag

pJJH124212609 bps

Nde I 1165


Not I 3529


RI 6660EcoI 7288Xho

URA3

CMV

CamKII

eGFPWPRE

f1 ori

SV40 ori

pUC ori

bla

2µm


YFGPCR product fromplasmid template withgene coupled to marker

C-terminal GFP tagged gene of interest

CamKII promoter

CamKII promoter

terminator

terminator




YFG

YFG

40 nt 20 nt

20 nt 40 nt

GFP


3‘

3‘

5‘

YFG -GFP

GFP

omit STOP-Codon in oligo design



2d. IVR cloning of genes of interest with N-terminal eGFP-tag

pJJH124212609 bps

Nde I 1165


Not I 3529


RI 6660EcoI 7288Xho

URA3

CMV

CamKII

eGFPWPRE

f1 ori

SV40 ori

pUC ori

bla

2µm

target vector linearized with EcoRI

YFGPCR product fromplasmid template withgene coupled to marker

N-terminal GFP-tagged gene of interest

CamKII promoter

CamKII promoter

terminator

terminator




YFG

YFG

40 nt 20 nt

20 nt 40 nt


GFP5‘

3‘

3‘

5‘

GFP-

GFPomit STOP-Codon in oligo design

YFG



3. Recovery of plasmids from yeast transformants

inoculate 3 ml selective medium with yeast transformant, grow ON at 30°C

harvest by centrifugation, wash, and resuspend in plasmid miniprep kit buffer 1

add glass beads and break cells by vigorous shaking (4°C/7 min)

centrifuge, collect supernatant and treat like E. coli plasmid preparation

elute from columns with 50 µl Tris-buffer, use 20 µl for E. coli transformationuse plasmid preparation and restriction digest for verification

Note: You cannot detect plasmids from a yeast preparation directly on an agarose gel!



4. Removal of yeast sequences in E. coli (optional)from: Bakota et al.

all yeast sequences can be removed, leaving a single loxP site in the target vector


Lecture overview

Shuttle vectors and use of yeast for in vivo cloning

Multiple deletions and tetrad analysis for combination of desired traits

Substitution of deletion cassettes for in vivo mutated genes

“Beyond the rim“: C- and N-terminal tags, yeast display, gap-repair, and many more


Example: A quintuple deletion of CWI sensor genes


Method: Homologous recombination at chromosomal loci

Recipient yeast strains (HD56-5A derivatives)MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP

GAL MAL3 SUC2MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP

GAL MAL3 SUC2


Method: Crossing and tetrad analysis

MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 wsc1::KlURA3

MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 mid2::KlLEU2

AgTEF2p AgTEF2t

wsc1::KlURA3AgTEF2p AgTEF2t

mid2::KlLEU2

Life cycle of laboratory S. cerevisiae strains with Mendelian inheritance

X

for example: MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 wsc1::KlURA3 mid2::KlLEU2

→ Tetrad analysis allows the combination of desired traits from different parental strains


Method: Crossing and tetrad analysis

-trp (or -his; or G418) -ura -leu

wsc2::KlTRP1wsc3::kanMXmtl1::SkHIS3

wsc1::KlURA3 mid2::KlLEU2

MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc1::KlURA3 wsc2::KlTRP1 wsc3::kanMXmtl1::SkHIS3 MID2

MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc2::KlTRP1 wsc3::kanMX mid2::KlLEU2 mtl1::SkHIS3 WSC1

X

homozygous deletions

heterozygous deletions

At least one of the sensors must be present for the yeastcell to be viable; i.e. the quintuple deletion is lethal


Regeneration of selection markers (e.g. for expression of human homologs)

MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc1::KlURA3 wsc2::KlTRP1 wsc3::kanMXmtl1::SkHIS3 MID2

AgTEF2p AgTEF2t

wsc1::KlURA3

AgTEF2p AgTEF2t

wsc2::KlTRP1

AgTEF2p AgTEF2t

wsc3::kanMX

AgTEF2p AgTEF2t

mtl1::SkHIS3

loxPloxP

MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc1::loxP wsc2::loxP wsc3::loxP mtl1::loxP MID2

wsc1::loxP

loxP

wsc2::loxP

wsc3::loxP

mtl1::loxP

pSH658018 bps

Stu I 917

Xho I 3363Sal I 3369

dIII 4779HinRI 4791Eco

TEF1p

ble

CYC1termCreGAL1p

ori

bla

CEN/ARS

Cre recombinasemediated excision

XV

XIV

XV

VII


SummaryTriple shuttle vectors allow for easy cloning of genes of interest in any vector of interest using in vivo-recombination in yeast

In vivo-recombination can also be efficiently used to delete any target sequence from the haploid (or diploid) yeast genome

Classical genetics with crossing and tetrad analysis can be employed to construct multiple deletions

Markers can be either exchanged for each other or can be regenerated using plasmid-based, induced Cre-recombinase expression


Method: Homologous recombination at chromosomal loci

AgTEF2p AgTEF2t

wsc1::KlURA3

AgTEF2p AgTEF2t

wsc2::KlTRP1

AgTEF2p AgTEF2t

wsc3::kanMX

AgTEF2p AgTEF2t

mid2::KlLEU2

AgTEF2p AgTEF2t

mtl1::SkHIS3

chrXV (YOR008C)

chrXIV (YNL283C)

chrXV (YOL105C)

chrXII (YLR332W)

chrVII (YGR023W)

AgTEF2p AgTEF2t

KlURA3

WSC1

AgTEF2p AgTEF2t

KlTRP1

WSC2

AgTEF2p AgTEF2t

kanMX

WSC3

In vivo recombination and yeast genetics as tools · 2017-02-28 · AG Genetik IRTG-Seminar - April...

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