Unit 2.1 Yeast Transformation and Cloning PDF

Yeast transformation. Yeast two- hybrid Dra. Macarena Rodríguez-Prados Valle 2024 By Mogana Das Murtey and Patchamuthu Ramasamy Macarena Rodríguez-Prados Valle Methods and...

Yeast transformation. Yeast two- hybrid Dra. Macarena Rodríguez-Prados Valle 2024 By Mogana Das Murtey and Patchamuthu Ramasamy Macarena Rodríguez-Prados Valle Methods and technics in biomedicine II Yeast transformation. Yeast two-hybrid Why yeast? Transformation conditions Plasmids lithium acetate technique Spheroplasts Biolistic Glass bead methods. Yeast two-hybrid Principles Technique Advantages Limitations Macarena Rodríguez-Prados Valle  2. Yeast transformation. Yeast two-hybrid 1. Master Class (9/10/24) 2. Master Class (16/10/23) -Why yeast? Yeast two-hybrid Transformation conditions Principles Plasmids Technique Advantages lithium acetate technique Limitations Spheroplast Biolistic -Audiovisual resources (Jove video) 2. Workshop (10/10/22) -Questions Presentations/questions/videos Macarena Rodríguez-Prados Valle Why yeast is a system required? - Yeasts are single cells and eukaryotic model system - Have short life cycle. - Genetically and physiologically well characterized Haploid genome has low complexity with size of nearly 12 Mbp. - They are fast and easy to growth - Many auxotrophic and other markers are known - A source of several strong promoters - Introduction of naked DNA is easy - Many yeast genes are functionally expressed in E.coli - They can be utilized effectively for protein production. Macarena Rodríguez-Prados Valle  Yeasts are prominent hosts for the production of recombinant proteins from industrial enzymes to therapeutic proteins. The similarity of protein secretion pathways between these unicellular eukaryotic microorganisms and higher eukaryotic organisms has made them a preferential host to produce secretory recombinant proteins. However, there are several bottlenecks, in terms of quality and quantity, restricting their use as secretory recombinant protein production hosts. Macarena Rodríguez-Prados Valle  Yeast used for protein production Saccharomyces cerevisiae Robust growth on simple media in large-scale bioreactors combined with their capacity for eukaryotic post-translational modifications and feasibility in genetic manipulations, yeasts are practical production hosts. Protein secretion involve numerous complex steps mediated by several hundred cellular proteins. Core secretion functions, including translocation through the ER membrane, primary glycosylation, folding and quality control and vesicle mediated secretion, are similar from yeasts to higher eukaryotes Macarena Rodríguez-Prados Valle Molecular and process tools available for bioprocess optimization doi: 10.3390/microorganisms7020040 Macarena Rodríguez-Prados Valle Macarena Rodríguez-Prados Valle  Limitations of yeast as expression systems However, despite several evident advantages of yeasts as host cells, there are certain limitations to their use as expression systems, including: 1- Inefficient secretion 2- improper folding 3- hyperglycosylation and 4- aberrant proteolytic processing of proteins. Several synthetic biological approaches have allowed the improvement of secretion efficiency and folding capacity, which affect the final yield and quality of recombinant proteins in the culture supernatant. Macarena Rodríguez-Prados Valle  Transformation conditions have been developed empirically Transformation yeast is when DNA will pass across the cell wall and plasma membrane of living cells, which are normally impermeable to DNA. Very few cells are naturally competent, or able to take up DNA on their own. Consequently, researchers use a variety of chemical treatments to render cells competent. These chemical treatments have some kind of destabilizing effect on the plasma membrane. The introduction of DNA into these competent cells can be further encouraged by a physical stress, such as a pulse of electric current or temperature elevation. - The structure of the DNA used for transformation greatly affects the transformation efficiency. Transformation efficiencies are considerably higher with supercoiled plasmid DNA than with linear pieces of DNA, possibly because plasmids enter the cell more readily and/or plasmids are less susceptible to endonuclease digestion. Macarena Rodríguez-Prados Valle The Plasmid Region Map Plasmids contain a multiple cloning site or MCS where restriction endonucleases, "restriction enzymes", can cut DNA. DNA fragments of interest cut with the same enzymes can then be ligated into the MCS. Plasmids also contain an origin of replication or ORI that signals to the cell where replication should begin. In addition, plasmids have a selectable marker, which allows the yeast cells that contain the plasmid to grow under specific environmental conditions. Yeast that don’t successfully incorporate the plasmid will not survive in media containing the selectable marker. The selectable markers can encode for genes that enable drug- resistance or genes that encode enzymes that enable a yeast strain to synthesize amino acids that they otherwise cannot produce. Macarena Rodríguez-Prados Valle Yeast artificial chromosome (YAC) Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC). Size matters: use of YACs, BACs and PACs in transgenic animals Macarena Rodríguez-Prados Valle DO - 10.1023/A:1008918913249 Yeast artificial chromosome (YAC) Size matters: use of YACs, BACs and PACs in transgenic animals DO - 10.1023/A:1008918913249 Macarena Rodríguez-Prados Valle Methods for yeast cells transformation Some of the common methods used in transformation of yeast cells are: - Lithium transformation - Electroporation - Spheroplasts - Biolistic - Glass bead methods. - These methods are commonly used for S. cerevisiae but can be used for transforming other fungi such as yeasts (e.g., Schizosaccharomyces pombe, Candida albicans and Pichiapastoris) and filamentous fungi (e.g., Aspergillus species). Macarena Rodríguez-Prados Valle REQUIREMENTS Transformation method involves three main steps: 1- Preparing competent yeast cells 2- Transformation with plasmid DNA 3- Subsequent plating to select the transformants. Review materials required and the detailed protocol of transformation. Macarena Rodríguez-Prados Valle How to prepare competent yeast for transformation? 1- Yeast cells must be prepared by first picking a colony from an agar plate and amplifying the colony in yeast extract peptone dextrose medium, abbreviated YPD - a complete medium for yeast growth. 2- After the colony is picked from a plate and placed into YPD medium, the culture is incubated overnight at 30 °C with agitation on a shaker or roller apparatus. 3- The yeast cells are pelleted by centrifugation and the supernatant is removed. The pelleted cells are resuspended with the desired buffer or sterile water. These competent prepared yeast cells will be used in the transformation procedure. 4- Once yeast cells have been prepared, transformation can be carried out by first preparing the transformation mixture. Transformation reagent mixture should include; sterile distilled water; a solution of 50% polyethylene glycol or PEG, 1M lithium acetate, 10 mg/ml solution of single-stranded DNA, plasmid DNA and competent yeast cells. The exact proportions of each solution should be calculated before beginning the experiment by consulting your laboratory’s standard protocol for yeast transformation. Macarena Rodríguez-Prados Valle Lithium Acetate transformation The mixture is then incubated at 30 °C for 30 minutes with shaking Transformation reagent mixture The cells are heat-shocked by placement in a 42 °C water bath for 15 minutes followed by cooling on ice for 2 minutes. Macarena Rodríguez-Prados Valle Transformation conditions have been developed empirically - The most used yeast transformation methods is a combination of lithium acetate, single- stranded carrier DNA and polyethylene glycol (PEG). - Lithium ions neutralize the negative charges on DNA molecules to be transformed and the phospholipid bilayer of the yeast cell, and they may also generate small holes in the plasma membrane that allow the passage of nucleic acids. - Single-stranded DNA acts as a carrier for the plasmid DNA to be transferred into the cell and it may help to protect the latter from endonucleases. - It is imperative that the carrier DNA for transformations be single-stranded. We will boil the carrier DNA for 5 minutes and then rapidly chill it to prevent reanneling of the DNA helix. - PEG may help bring the DNA into closer apposition with the membrane. PEG is often used to promote membrane fusion and is thought to alter water structure around plasma membranes Macarena Rodríguez-Prados Valle  Lithium acetate procedure Cell membrane and Positive charged Single strain of DNA The heat shock plasmid DNA are Lithium cation added to the creates a naturally negatively neutralize the negative transformation pressure charged. charges in both cell mixture bind to the cell between the membrane and wall of the yeast and inside and plasmid DNA. leave the plasmid DNA outside of the available for uptake for cell creating the yeast cell. pores. Macarena Rodríguez-Prados Valle Yeast Lithium Transformation 1. Prepare YPD and synthetic complete (SC) drop-out medium plates and autoclave them separately. 2. Inoculate yeast cells from plates into 20 mL of YPD medium in a 100 mL sterile flask. 3. Grow overnight with shaking. 4. Dilute cells from above culture into 100 mL of YPD medium until the OD600 is 0.3 5.Pellet cells gently. 6.Resuspend in 7-8 mL of 1x TE-LiAc solution and rotate at 23 °C for 1-1.5 hours. Macarena Rodríguez-Prados Valle 7. Add 10 µL of 10 mg/mL salmon testes DNA (Catalog Number D9156) in sterile microfuge tubes designated for transformation and one for a negative control. 8. Add 0.1 µg of yeast plasmid DNA (to be studied) to each tube and 100 µL of competent cells into each tube and then vortex. 9. Add 600 µL of freshly prepared PEG-TE-LiAc solution, vortex, and incubate at 30 °C for 30 minutes with shaking. 10. Optional - DMSO (Catalog Number D8418) can be added to 10% (v/v); followed by heat shock for 15 minutes at 42 °C. 11. Spin for 3 seconds, resuspend cells in sterile water and plate using appropriate SC drop-out medium. Macarena Rodríguez-Prados Valle Macarena Rodríguez-Prados Valle Links of interest: Video 1: Yeast Transformation and Cloning https://www.jove.com/es/embed/player?id=5083&access=l780ryv6h3&t=1&s=1&fpv=1 Video 2: Isolating Nucleic Acids from Yeast https://www.jove.com/v/5096/isolating-nucleic-acids-from-yeast https://www.jove.com/v/5096/isolating-nucleic-acids-from-yeast Video 3: Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice https://www.jove.com/es/v/10515/transformation-e-coli-cells-using-an-adapted-calcium-chloride?list=h00KUa1V https://www.jove.com/es/v/10515/transformation-e-coli-cells-using-an-adapted-calcium-chloride?list=h00KUa1V Video 4: Biolistic BIO-RAD tutorial https://www.youtube.com/watch?v=dfD95gsEdrg&t=16s Macarena Rodríguez-Prados Valle Spheroplasts What is a Spheroplast? A Spheroplast can be described as a microbe organism with a cell wall that is almost entirely gone through the penicillin or Lysozyme. The term is employed to refer to Gram-negative bacteria and also includes yeasts. Spheroplast’s name is derived in the sense that once the cell wall of the microbe is digested by membrane tension, the cell to take on the characteristic shape of a sphere. Spheroplasts can be osmotically fragile and can lyse when transferred to a hypotonic environment. In the context of Gram-negative bacteria ”spheroplast” refers to cells in which the peptidoglycan part but not the membrane that forms the cell wall has been removed. Macarena Rodríguez-Prados Valle Macarena Rodríguez-Prados Valle Spheroplast method transformation Lyticase also known as Zymolyase is an enzyme mixture used to degrade the cell wall of yeast and form spheroplasts. 1. Grow the cells 2. Wash the cells 3. Digest the cell wall with zymolyase 4. Harvest the spheroplasts and mix with plasmid DNA 5. Resuspend the spheroplasts in 1 M sorbitol 6. Incubation for 3–4 days at 30°C. Basic procedures in yeast genetic engineering. Reproduced from Walker GM (1998). Yeast Physiology and Biotechnology. Chichester, UK: John Wiley & Macarena Rodríguez-PradosSons. Valle  Protocol for the spheroplast method The removal of the yeast cell wall by enzymatic treatment to yield protoplasts is essential to promote the transformation. Basic procedures in yeast genetic engineering. Reproduced from Walker GM (1998). 1. Cells are grown in 50 mL YPAD to a density of 3 × 107 cells/mL. 2. The cells are harvested by centrifugation at 400–600× g for 5 min, washed twice in 20 mL sterile water, and washed once in 20 mL 1 M sorbitol. The cells are resuspend in 20 mL SPEM (1 M sorbitol, 10 mM sodium phosphate, pH 7.5, 10 mM EDTA plus 40 µL β- mercaptoethanol added immediately before use). 3. The cells are converted to spheroplasts by the addition of 45 µL zymolyase 20T (10 µg/mL) and incubation at 30°C for 20–30 min with gentle shaking. By this time, 90% of the cells should be converted to spheroplasts. R. Daniel Gietz and Robin A. Woods, 2001. Genetic Transformation of Yeast Macarena Rodríguez-Prados Valle  Protocol for the spheroplast method 4. The spheroplasts are harvested by centrifugation at 250× g for 4 min, and the supernatant is removed carefully. The pellet is washed once in 20 mL STC (1 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl2 ) and resuspended in 2 mL STC. 5. Spheroplasts are transformed by gently mixing 150 µL of the suspension in STC with 5 µg carrier DNA and up to 5 µg plasmid DNA in less than 10 µL. The mixture is incubated for 10 min at room temperature. One milliliter of PEG reagent [10 mM Tris-HCl, pH 7.5, 10 mM CaCl2 , 20% (w/v) PEG 8000; filter sterile] is added and mixed gently, and incubation is continued for another 10 min. 6. The spheroplasts are harvested by centrifugation for 4 min at 250× g and resuspended in 150 µL SOS (1.0 M sorbitol, 6.5 mM CaCl2 , 0.25% yeast extract, 0.5% bactopeptone). Dilutions of spheroplasts are mixed with 8 mL TOP (selective medium containing 1.0 M sorbitol and 2.5% agar kept at 45°C) onto the appropriate selective medium containing 0.9 M sorbitol and 3% glucose. Transformants can be recovered after incubation for 3–4 days at 30°C. Macarena Rodríguez-Prados Valle Spheroplasts Conversion of Schizosaccharomyces pombe cells to spheroplasts using Novozym 234, a commercially available enzyme preparation. Macarena Rodríguez-Prados Valle DICKINSON and ISENBERG 1982 - Metal microprojectiles coated with nucleic acid can be shot into cells with consequent expression of the introduced genes (Klein et al. 1987). - This "biolistic" (biological-ballistic) transformation methodology was developed specifically to deliver nucleic acid through the wall of intact plant cells in situ (Sanford et al. 1987). - Transient expression of biolistically transferred genes has been demonstrated with several mono and dicotyledonous plants including corn, tobacco, rice, wheat, and soybean (Klein et al. 1988a, b, c; Wang Macarena et al. 1988). Rodríguez-Prados Valle - Among the physical factors affecting the efficiency of the process in yeast are the microprojectile's constitution, size, concentration and amount, and the procedure used for binding DNA to it. Tungsten microprojectiles coated with nucleic acid and accelerated to velocities of approximately 500 m/s, can penetrate living cells and tissues with consequent expression of the introduced genes (Klein et al. 1987). - The biological parameters that affect the process include the cell's genotype, growth phase, plating density, and the osmotic composition of the medium during bombardment. Saccharomyces cerevisiae is used here as a model system to define the basic parameters governing the biolistic (biological-ballistic) delivery of DNA into cells. - By optimizing these physical and biological parameters, rates of transformation between 10 -5 and 10 -4 were achieved. Stable nuclear transformants result primarily from penetration of single particles of 0.5- 0.65 gm in diameter, delivering on average 10-30 biologically active plasmids into the cell. The tungsten particles detectably increase the buoyant density of the transformants' progenitors. Macarena Rodríguez-Prados Valle Performing a Bombardment Quick Guide Before the Bombardment 1. Select/adjust bombardment parameters for Gap distance between rupture disk retaining cap and microcarrier launch assembly. Placement of stopping screen support in proper position inside fixed nest of microcarrier launch assembly 2. Check helium supply (200 psi in excess of desired rupture pressure). 3. Clean/sterilize: Equipment: rupture disk retaining cap, microcarrier launch assembly Consumables: macrocarriers/macrocarrier holders 4. Wash microcarriers and resuspend in 50% glycerol 5. Coat microcarriers with DNA and load onto sterile macrocarrier/macrocarrier holder the day of experiment Firing the Device 1. Plug in power cord from main unit to electrical outlet. 2. Power ON. 3. Sterilize chamber walls with 70% ethanol. 4. Load sterile rupture disk into sterile retaining cap. (Rupture can be immersed in iso-propanol when using) 5. Secure retaining cap to end of gas acceleration tube (inside, top of bombardment chamber) and tighten with torque wrench. 6. Load macrocarrier and stopping screen into microcarrier launch assembly. 7. Place microcarrier launch assembly and target cells in chamber and close door. 8. Evacuate chamber, hold vacuum at desired level (minimum 5 inches of mercury). 9. Bombard sample: Fire button continuously depressed until rupture disk bursts and helium pressure gauge drops to zero. 10. Release Fire button. After the Bombardment 1. Release vacuum from chamber. 2. Target cells removed from chamber. 3. Unload macrocarrier and stopping screen from microcarrier launch assembly. 4. Unload spent rupture disk. 5. Remove helium pressure from the system (after all experiments completed for the day). Macarena Rodríguez-Prados Valle https://www.youtube.com/watch?v=dfD95gsEdrg&t=16s  Photos, figures and tables are from www.bio-rad.com website. Macarena Rodríguez-Prados Valle Procedures were developed by Sanford et al (1992)  Photos, figures and tables are from www.bio-rad.com website. Macarena Rodríguez-Prados Valle Procedures were developed by Sanford et al (1992) Macarena Rodríguez-Prados Valle R. Daniel Gietz and Robin A. Woods, 2001.  Selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes (An antibiotic resistance marker is a gene that produces a protein that provides cells expressing this protein with resistance to an antibiotic. Positive or selection markers are selectable markers that confer selective advantage to the host organism. An example would be antibiotic resistance, which allows the host organism to survive antibiotic selection. Negative or counter selectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example would be thymidine kinase, which makes the host sensitive to ganciclovir selection. Positive and negative selectable markers can serve as both a positive and a negative marker by conferring an advantage to the host under one condition, but inhibits growth under a different condition. An example would be an enzyme that can complement an auxotrophy (positive selection) and be able to convert a chemical to a toxic compound (negative selection). Macarena Rodríguez-Prados Valle Examples of selectable markers include: - Beta-lactamase which confers ampicillin resistance to bacterial hosts. - Neo gene from Tn5, which confers resistance to kanamycin in bacteria and geneticin in eukaryotic cells. - Mutant FabI gene (mFabI) from E. coli genome, which confers triclosan resistance to the host. - URA3, an orotidine-5' phosphate decarboxylase from yeast is a positive and negative selectable marker. It is required for uracil biosynthesis and combined with ura3 mutants yeast that lacks the URA3 allele allow them to survive (positive selection). The enzyme URA3 also converts 5-fluoroorotic acid (5FOA) into the toxic compound 5-fluorouracil, so any cells carrying the URA3 gene will be killed in the presence of 5FOA (negative selection). Macarena Rodríguez-Prados Valle Links of interest: Video 1: Yeast Transformation and Cloning https://www.jove.com/es/embed/player?id=5083&access=l780ryv6h3&t=1&s=1&fpv=1 Video 2: Isolating Nucleic Acids from Yeast https://www.jove.com/v/5096/isolating-nucleic-acids-from-yeast https://www.jove.com/v/5096/isolating-nucleic-acids-from-yeast Video 3: Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice https://www.jove.com/es/v/10515/transformation-e-coli-cells-using-an-adapted-calcium-chloride?list=h00KUa1V https://www.jove.com/es/v/10515/transformation-e-coli-cells-using-an-adapted-calcium-chloride?list=h00KUa1V Video 4: Biolistic BIO-RAD tutorial https://www.youtube.com/watch?v=dfD95gsEdrg&t=16s Macarena Rodríguez-Prados Valle  References Principles and Techniques of Biochemistry and Molecular Biology Seventh edition EDITED BY KEITH WILSON AND JOHN WALKER, 2010. Genetic Transformation of Yeast Lin JS, Lai EM (2017) Protein-Protein Interactions: Yeast Two-Hybrid System.doi: 10.1007/978-1-4939-7033-9_14 DICKINSON and ISENBERG (1982) Preparation of Spheroplasts. DOI:10.1099/00221287-128-3-651 Size matters: use of YACs, BACs and PACs in transgenic animals DOi - 10.1023/A:1008918913249 Armaleo et al., (1989). Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi. doi: 10.1007/BF00312852. High-throughput Yeast Plasmid Overexpression Screen https://www.jove.com/es/v/2836/high-throughput-yeast-plasmid-overexpression-screen Macarena Rodríguez-Prados Valle Questions? Macarena Rodríguez-Prados Valle  Flipped Class-Oral presentations 25 min per group Topics 1-Transformation of Intact yeast cells treated with Alkali Cations 2-Extremely simple, rapid and highly efficient transformation 3-Biolistic nuclear transformation Presentation -Introduction: Basic principles -Material and methods -Procedure -Conclusn Macarena Rodríguez-Prados Valle