Presentation on theme: "Molecular biology tools for the genetic manipulation of yeast Plasmids, transformation and working the numbers."— Presentation transcript:
Molecular biology tools for the genetic manipulation of yeast Plasmids, transformation and working the numbers
Screen vs. Selection Screen: ALL colonies are looked at, we chose the ones with the right phenotype (e.g. expressing galactosidase in a screen designed to identify mutants in a certain regulatory pathway we pick the blue colonies) Selection: Only the colonies we are interested in will survive (e.g. selecting for transformants)
Suppression vs. complementation Definition of complementation: The production of a wild-type phenotype by re-introduction of the wild type gene into the mutant (either by plasmid transformation or mating with a strain that carries the wild type copy of the gene). –Complementation also by introduction of a functional homologue A suppressor is generally defined as a mutation that completely or partially restores the mutant phenotype of another mutation –Multicopy suppression: overexpression of one gene can completely or partially restore the mutant phenotype of another mutation
Yeast markers Marker definition: A) allele of a gene that allows identification of the strain itself e.g. Mutations (“marker mutation”) in biosynthetic pathways (in a strain: mutations in ade2,ura3, leu2, trp1….) Example: Yeast strain W1536 5B: MATa; ade2Δ; ade3Δ; leu2-3; his3-11; his3-12; trp1-1; ura3-1 (Rothstein W303 derivative)
Yeast markers B) Gene on a plasmid/piece of DNA that allows for the identification of a plasmid in the cell a gene that confers a certain ability to the strain : ADE2, URA3, LEU2, TRP1, KanMX = geneticin resistance
Plasmids/Vectors What is a Plasmid? What is a Vector?
Genomic DNA Library Collection of plasmids/vectors carrying pieces of genomic DNA from your organism of choice Nucleus with DNA Isolate DNA Cut into little pieces with restriction enzymes Ligate all different pieces into vector Collection of plasmids representing the entire genome (or rather a large fraction thereof) Genomic library: cDNA library: containing DNA fragments reverse transcribed from mRNA
Number of fragments of genomic DNA required to be cloned into vector to cover entire genome ln (1-P) ln (1-F) N= N: number of library members required P: Probability that gene is in library F: Average size of Fragments Size of genome Example: yeast about 10 4 kb; choose P=99%, 15 kb fragment size ln (1-0.99) Ln (1- ) 15 10 4 ~ 3070 fragments/library members required “complexity of the library”
Requirements for plasmids and vectors Plasmid: Vector:
1.Finding a nutritional marker (e.g. LEU2 involved in leucine biosynthesis)
Generation of genetic tools: How to find the ingredients to make a yeast vector
Selectable marker: Cloning of a yeast gene involved in Leucine biosynthesis Cells are Leu - (cannot grow on media lacking Leucine) LEU2 ori bla Transform E. coli leuB- mutants with library containing yeast DNA Select for bacterial transformants on minimal media (lacking Leucine) LEU2 ori bla gene W ori bla geneY ori bla geneX ori bla E. coli cell, with leuB mutation Isolate plasmid High frequency transformation! Cloning by functional complementation
LEU2 ori bla Transform Plasmid into Leu - strain Select for yeast transformants on synthetic media lacking Leucine Low frequency transformation! Recombination event required! leu2- ori bla LEU2 leu2-LEU2 ori bla leu2- INEFFICIENT! Integrating Vectors) SC-Leu
leu2- ori bla LEU2 leu2- ori bla leu2- ori bla LEU2 ori bla leu2- + Double Single Recombination event Reversible! (somewhat unstable) “Popout” event LEU2 Leu2- Irreversible! (very stable) Transformation possible but very low frequency/ inefficient
2. Finding an origin of replication (ARS = automomously replicating sequence)
Cloning of ARS fragment LEU2 ori bla Yeast genomic DNA (make yeast genomic DNA library) leu2- Transform yeast Low efficiency Pick transformants (keep a stock) Grow non-selectively (e.g. on YPD) for several generations SC-Leu
Plate cells from non-selective culture on Non-selective plate (e.g. YPD) Replica plate on selective culture (SCD– Leu) Leu+ - marker lost at high frequency -> plasmid- borne (not integrated in genome)! Isolate DNA
Re-transform LEU2 ori bla ARS leu2- Transform yeast SC-Leu High frequency of transformation! But: Unstable (Lost at high frequency Need centromeric function on plasmid
3. Isolation of a CEN sequence
Make new yeast library with marker gene and ARS + genomic DNA insert LEU2 ori bla leu2- ARS Yeast genomic DNA Transform SC-Leu High frequency of transformation (collect large number of colonies)
Grow non-selectively (e.g. YPD) will enrich for stable transformants ( which have CEN fragment) other cells will lose plasmid if without centromeric sequence 2828 2828 2828 2828 8888 Transformant with stable plasmid: 20% of cells After 3 doublings Transformant with stable plasmid: 50% of cells viable on SC - Leu Enrichment for cells containing stable plasmid isolate after large number of generations growing non-selectively! (Plate cells on SC- Leu) Isolate plasmids, retransform to confirm stability, sequence…
Yeast Plasmids CEN based vectors: containing yeast centromeric fragment and yeast ARS, relatively stable (lost~5-10% every generation) centromere is important for – Proper segregation –Control of replication (only once during cell cycle) 2 minicircle – based (naturally occuring plasmid in yeast cir+ cells) multicopy (10-50 copies per cell), relatively stable (lost at about 5-10% per generation) Integrating Vectors (low trafo frequency, but very stable when integrated
TEL: The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases. CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell. ORI: Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks. It also contains few other specific sequences like: A and B: selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome. Recognition site for the two restriction enzymes EcoRI and BamHI. YACs (Yeast Artificial Chromosomes )
- While DNA cloning into a plasmid allows the insertion of DNA fragment of about 10,000 nucleotide base pairs, DNA cloning into a YAC allows the insertion of DNA fragments up to 1,000,000 nucleotide base pairs. - Why is it so important to be able to clone such large sequences? To map the entire human genome (3x1,000,000,000 nucleotide base pairs) it would require more than 1,000,000 plasmid clones. In principle, the human genome could be represented in about 10,000 YAC clones. - YACs can be isolated in their full size by pulsed field gel electrophoresis (PFGE) - Techniques for cloning genomic DNA into yeast artificial chromosomes (YAC) make it possible to analyze very long DNA sequences like human genes
Yeast transformation: Usually done in PEG/DTT/LiAcetate, heat shock (45 o C; 30-40 minutes) Simple one-step procedure (10 2 – 10 4 transformants per g of DNA) (45 minutes) Or procedure involving growth steps and transformation at a specific growth stage (10 4 -10 6 transformants per g of DNA) (half a day)
Generating gene knockouts in yeast
YIp plasmids were used for gene knockouts/replacements in yeast (2-step gene replacement) FOA (5-fluoro-orotic acid) kills cells that carry a functonal URA3 - gene selection for plasmid “popout”
YFG1 URA3 YFG1 ori bla Yfg1-1 yfg1-1 ori bla YFG1 + Double Single Recombination event Reversible! yfg1-1 Yfg1-1 Irreversible Transformation possible but very low frequency/ inefficient URA3 yfg1-1 YFG1 URA3 YFG1
One-step gene replacement: -Uses a marker gene with flanking sequences homologous to the gene of interest -Can be generated by PCR: KANMX ori bla YFG 5’YFG 3’ YFG 5’YFG 3’ KANMX YFG 5’YFG 3’ KANMX YFG yfg::KANMX Integration via homologous recombination PCR reaction Select for geneticin resistance
Isolate yeast DNA from transformants - Verify integration by PCR yfg::KANMX YFG Product 1.7kb 1.3 kb 1. 2. 3. Product (1.2 kb)No product Product (0.8 kb) No product About 2-3 weeks to create and confirm a yeast knockout! How much in Mouse?
Plasmid shuffle: Done when working with essential gene: Phenotype of mutant versions of the gene can be investigated in a deletion background
Introduction into the Diploid and tetrad dissection/selection of the haploid transformant RFT1 rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- sporulate
4 spores of a tetrad rft1::KANMX RFT1 ura3- CEN RFT1 URA 3 p GAL 1 t CY C1 CEN rft1 URA 3 p GAL 1 t CY C1 Dissect!
rft1::KANMX RFT1 ura3- CEN RFT1 URA 3 p GAL 1 t CY C1 CEN RFT1 URA 3 p GAL 1 t CY C1 Dissect on YPGalactose!!!! rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- DEAD! RFT1 CEN RFT1 URA3 p GAL1 t CYC1 ura3- eitheror
RFT1 CEN RFT1 URA3 p GAL1 t CYC1 ura3- rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- Select cells that carry the knockout allele plus plasmid RFT1 ura3- A. Select on SC – ura (Galactose) Can grow DEAD!
rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- RFT1 CEN RFT1 URA3 p GAL1 t CYC1 ura3- B. Select for growth on YPGalactose - geneticin Can grow DEAD!
rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- RFT1 CEN RFT1 URA3 p GAL1 t CYC1 ura3- C. Test Growth on SC-Galactose – FOA (5-fluoro-orotic acid) Can grow (can lose plasmid) DEAD! (cannot lose plasmid)
rft1::KANMX CEN RFT1 URA3 p GAL1 t CYC1 ura3- Perform experiments with isolate 1.Grow on Raffinose – switch to glucose repression of RFT1 phenotype of Rft1p depletion can be studied 2. Grow on Raffinose – switch to galactose activation of RFT1 phenotype of Rft1p overexpression can be studied
Reporter genes Reporter genes are heterologous genes that confer and easily detectable phenotype to the object of study Reporter genes are often used in the analysis of gene expression -examples: - galactosidase (E. coli lacZ gene): can be assayed quantitatively (cleavage of ONPG) cleaves X-gal to produce blue colour (visual assay) Use: for example identify genes involved in repression by glucose; fuse promoter of glucose-repressed gene to - galactosidase; mutagenize screen for blue colonies on glucose = yeast cells mutant for glucose repression Reporter Gene ( e.g lacZ) Promoter of YFG >Transcription
Reporter genes (continued) examples: -HIS3 (involved in Histidine biosythesis) can be competitively inhibited by 3-amino-triazole can be used in selection for viability on media lacking histidine; especially used to detect strong activating activity of transcription factors (e.g. in two-hybrid screens) -CAT (chloramphenicol acetyl transferase) Transfers radioactive acetyl groups to chloramphenicol; detection by thin layer chromatography and autoradiography