IRTG-Seminar - April 25, 2012
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AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch In vivo recombination and yeast genetics as tools for studying microcompartmentation IRTG-Lecture Jürgen J. Heinisch April 25, 2012 AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch 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 AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch 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 IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch Characteristics of shuttle vectors (2-way/double) - maintenance and expression in YFO (= “Your Favorite Organism“) - suitable cloning sites - selection and amplification in E. coli Yeast/E. coli Eco RI 230 Sma I 246 Bam HI 251 Xba I 257 Sal I 263 Sph I 275 E. coli replication multiple cloning site lacZα bla Bcl I 847 Nhe I 891 URA3 1000 YEp352 4000 5407 bps Sca I 3910 2000 E. coli selection Spe I 252 Nde I 486 Sca I 9545 E. coli replication ori 5000 Lentivirus/E. coli CMV bla LTR5' Psi ori Nde I 1206 8000 yeast selection L22_eGFP 2000 9987 bps SV40 ori 6000 4000 CamKII 3000 f1 ori E. coli selection LTR3' WPRE 2µm yeast replication mammalian propagation Xho I 5283 eGFP expression signals Bam HI cloning site YFG Eco RI 4655 (= Your Favorite Gene) AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch 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 BamHI BglII BclI YFG-ORF - restriction site composition may interfere with translation efficiency of mRNA generated from a cDNA clone XmaI mRNA Protein ------ C C C G G G AUG -----Met – Aa – Aa – Aa ------ IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch The solution: Cloning by in vivo recombination (IVR) The principle: target sequence linearized with BamHI Spe I 252 NdeI 486 Sca I 9545 terminator CamKII promoter CMV bla LTR5'Psi ori 8000 L22_eGFP YFG 2000 9987 bps PCR product SV40 ori 6000 4000 CamKII f1 ori LTR3' WPRE Xho I 5283 eGFP Bam HI EcoRI 4655 CamKII promoter YFG terminator gene cloned into expression vector Yeast, Saccharomyces cerevisiae, is the best tool for IVR cloning (fast, efficient, accurate) IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch 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 EcoRI 230 SmaI 246 BamHI 251 XbaI 257 SalI 263 SphI 275 Oligo 2 300 bp lacZα 5000 YEp352 4000 5407 pUC ori 2µm BclI 847 NheI 891 URA3 bla ScaI 3910 URA3 bla 5‘ ori bla 2µm 1000 bps Nde I 1165 NdeI 1206 URA3 Spe I 2257 Nde I 2491 12000 Spe I 252 NdeI 486 Sca I 9545 2000 3000 2000 10000 Oligo 1 CMV CMV pJJH1242 2µm bla LTR5'Psi ori 12609 bps 4000 SV40 ori Not I 3529 8000 8000 f1 ori 6000 L22_eGFP 2000 9987 bps SV40 ori WPRE eGFP CamKII LTR3' Xho I 7288 Eco RI 6660 Not I 6645 6000 4000 CamKII f1 ori WPRE eGFP Bam HI Bam HI 5899 Xho I 5283 EcoRI 4655 40 bp IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 1b. Yeast sequences for Drosophila vectors 40 bp Oligo 1 PstI 35 Sph I 1 Sal I 41 Eco RI 380 XbaI 95 Eco RV 10953 StuI 571 loxP BamHI 1278 NcoI 800 EcoRV 819 UAS-HSPp URA3 Nsi I 9990 5000 bla 2µm 4000 loxP Sph I 1 pJJH1396 Bam HI 9039 1000 2000 URA3 Bam HI 1278 2000 white-SV40 117622µm bps loxP UAS-HSPp pJJH1410 3000 Nsi I 8423 Eco RI 380 Bgl II 392 Not I 398 Nde I 8264 512210000 bps Eco RV 9060 loxP loxP Bgl II 392 Not I 398 Bam HI 11751 40 bp 2µm URA3 bla 8000 8000 4000 loxP pUAST 6000 2000 9050 bps bla Eco RV 2712 6000 white-SV40 4000 Oligo 2 Eco RV 4279 Nsi I 5317 Nsi I 5317 Eco RV 4279 IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 1c. Yeast sequences for insect vectors Xba I 4678 Not I 4663 Spe I 4656 Sal I 4644 Eco RI 4628 BamHI 4606 Xho I 4311 Nhe I 4298 Nsi I 4287 Sph I 4280 Bam HI 1 Eco RI 23 Sal I 39 Not I 58 Pst I 81 Xho I 8343 Sph I 7602 Kpn I 7596 SV40PolyA P10/PHp PHp P10 5000 bla 1000 pFastBac dual 8000 4000 5238 bps bla pJJH1460 2000 2000 Gentamycin 3000 6000 Gentamycin ori 8637 bps Eco RV 6245 Eco RV 6117 ori loxP 4000 Eco RV 2923 2µm loxP URA3 Pst I 3440 Sal I 3446 Eco RV 4224 IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 2a. IVR cloning of genes of interest coupled to a selectable marker target vector linearized with BamHI 2µm bla URA3 pUC ori Spe I 2257 Nde I 2491 CMV pJJH1242 12609 bps SV40 ori Not I 3529 f1 ori CamKII WPRE eGFP Xho I 7288 Eco RI 6660 Bam HI 5899 Not I 6645 Oligonucleotides for PCR reaction: 5‘ 40 nt target vector (5‘) 3‘ 20 nt marker terminator CamKII promoter Nde I 1165 20 nt YFG PCR product from plasmid template with gene coupled to marker YFG marker e.g. kanMX (Geneticin) KlLEU2 SkHIS3 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) 3‘ 40 nt target vector (3‘) terminator marker gene cloned into expression vector CamKII promoter 5‘ YFG IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 2b. IVR cloning of genes of interest directly from cDNA target vector linearized with BamHI 2µm bla URA3 pUC ori Spe I 2257 Nde I 2491 CMV pJJH1242 12609 bps SV40 ori Not I 3529 f1 ori CamKII WPRE eGFP Xho I 7288 Eco RI 6660 Bam HI 5899 Not I 6645 Oligonucleotides for PCR reaction: 5‘ 40 nt target vector (5‘) 3‘ 20 nt YFG terminator CamKII promoter Nde I 1165 20 nt YFG PCR product from plasmid template with gene coupled to marker YFG 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) 3‘ 40 nt target vector (3‘) CamKII promoter 5‘ YFG terminator gene cloned into expression vector IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 2c. IVR cloning of genes of interest with C-terminal eGFP-tag target vector linearized with BamHI bla URA3 pUC ori CamKII promoter Nde I 1165 2µm CMV 12609 bps SV40 ori Not I 3529 f1 ori CamKII WPRE eGFP Bam HI 5899 Not I 6645 Oligonucleotides for PCR reaction: 5‘ 40 nt target vector (5‘) 3‘ 20 nt YFG 20 nt YFG 40 nt GFP terminator Spe I 2257 Nde I 2491 pJJH1242 Xho I 7288 Eco RI 6660 GFP PCR product from plasmid template with gene coupled to marker YFG omit STOP-Codon in oligo design 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) 3‘ CamKII promoter 5‘ YFG -GFP terminator C-terminal GFP tagged gene of interest IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 2d. IVR cloning of genes of interest with N-terminal eGFP-tag target vector linearized with EcoRI CamKII promoter Nde I 1165 2µm bla URA3 pUC ori CMV 12609 bps SV40 ori Not I 3529 f1 ori CamKII WPRE eGFP Bam HI 5899 Not I 6645 Oligonucleotides for PCR reaction: 5‘ 3‘ 40 nt 20 nt GFP YFG 20 nt YFG 3‘ 40 nt target vector (3‘) PCR product from plasmid template with gene coupled to marker YFG Co-transformation of linearized (mandatory) vector (10-50 ng) with PCR product (5-10 µg) into S. cerevisiae CamKII promoter 5‘ terminator omit STOP-Codon in oligo design Spe I 2257 Nde I 2491 pJJH1242 Xho I 7288 Eco RI 6660 GFP Selection for vector (on plates lacking uracil) GFP- YFG terminator N-terminal GFP-tagged gene of interest AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 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 transformation use plasmid preparation and restriction digest for verification Note: You cannot detect plasmids from a yeast preparation directly on an agarose gel! AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch A step-by-step procedure for the construction and use of triple shuttle vectors 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 AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch 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 AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch Example: A quintuple deletion of CWI sensor genes AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch 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 SUC2 MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch Method: Crossing and tetrad analysis Life cycle of laboratory S. cerevisiae strains with Mendelian inheritance MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 wsc1::KlURA3 wsc1::KlURA3 AgTEF2p AgTEF2t X MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP GAL MAL3 SUC2 mid2::KlLEU2 mid2::KlLEU2 AgTEF2p AgTEF2t 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 IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch Method: Crossing and tetrad analysis MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc1::KlURA3 wsc2::KlTRP1 wsc3::kanMX mtl1::SkHIS3 MID2 -trp (or -his; or G418) wsc2::KlTRP1 wsc3::kanMX mtl1::SkHIS3 homozygous deletions X MATα ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc2::KlTRP1 wsc3::kanMX mid2::KlLEU2 mtl1::SkHIS3 WSC1 -ura -leu wsc1::KlURA3 mid2::KlLEU2 heterozygous deletions At least one of the sensors must be present for the yeast cell to be viable; i.e. the quintuple deletion is lethal IRTG-Seminar - April 25, 2012 AG Genetik Jürgen J. Heinisch 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::kanMX mtl1::SkHIS3 MID2 Stu I 917 CEN/ARS wsc1::KlURA3 AgTEF2p bla pSH65 AgTEF2t 8018 bps ori loxP loxP wsc2::KlTRP1 AgTEF2p Cre recombinase mediated excision ble GAL1p Cre CYC1term Eco RI 4791 Hin dIII 4779 AgTEF2t TEF1p Xho I 3363 Sal I 3369 wsc3::kanMX AgTEF2p AgTEF2t mtl1::SkHIS3 AgTEF2p AgTEF2t wsc1::loxP XV wsc2::loxP XIV wsc3::loxP XV mtl1::loxP VII MATa ura3-52 leu2-3,112 his3-11,15 trp1N::loxP wsc1::loxP wsc2::loxP wsc3::loxP mtl1::loxP MID2 loxP AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch Summary Triple 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 plasmidbased, induced Cre-recombinase expression AG Genetik IRTG-Seminar - April 25, 2012 Jürgen J. Heinisch Method: Homologous recombination at chromosomal loci KlURA3 wsc1::KlURA3 AgTEF2t AgTEF2p chrXV (YOR008C) WSC1 AgTEF2p AgTEF2t wsc2::KlTRP1 chrXIV (YNL283C) KlTRP1 chrXV (YOL105C) WSC2 AgTEF2t wsc3::kanMX AgTEF2t AgTEF2p AgTEF2p AgTEF2p AgTEF2t mid2::KlLEU2 chrXII (YLR332W) kanMX AgTEF2p AgTEF2t mtl1::SkHIS3 AgTEF2t chrVII (YGR023W) WSC3 AgTEF2p AgTEF2p AgTEF2t