Directed Evolution 2 - BL5602-L5 PDF

Evolution - The only theory in Biology The theory of evolution states that the diverse array of organisms on Earth has evolved from a single common ancestor...

Evolution - The only theory in Biology The theory of evolution states that the diverse array of organisms on Earth has evolved from a single common ancestor over time. Evolution works through a process known as natural selection. Natural selection makes it more likely for the best-adapted individuals to survive and reproduce. These beneficial traits are then passed to the next generation. Over generations, natural selection of inherited traits could give rise to new species. Main differences The wild cabbage between natural and artificial selection The agent of selection man (breeder) versus nature (environment) The traits being selected Natural selection produces a great biological diversity. Artificial selection facilitates the inheritance of desired characters by the breeder. Chickens (on same diet) 1957 1977 2005 0 days The same strain 28 days was selected up to the date shown 56 days M. J. Zuidhof et al. Poultry Science 2014;ps.2014-04291  Growth hormone transgenic fish Activate better when it is Cold promoter - tiFreering opAFP-sGH chimeric gene construct Salmon Growth Hormone gene Ocean pout AFP Ocean pout AFP Promoter termination signal AFP=antifreeze protein gene 办 Nof normally expressed Prof. Hew Choy Leong under low temperature. former head of DBS Atlantic Salmon 伞 continue to transgenic Fish can winter grow during Du, S.J., Z. Gong, G.L. Fletcher, M.A. Shears, M.J. King, D.R. Idler, and C.L. Hew (1992) Growth enhancement in transgenic Atlantic salmon by use of fish antifreeze/growth hormone chimeric gene constructs. Bio/Technology 10:176- 181.  Growth hormone transgenic fish opAFP-sGH chimeric gene construct Ocean pout AFP Salmon Growth Hormone gene Ocean pout AFP Promoter termination signal AFP=antifreeze protein gene Prof. Hew Choy Leong former head of DBS 2000 Spin out Aquabounty for transgenic salmon 2003 Submitted to FDA for regulatory studies 2006 Aquabounty listed in London Stock Exchange Alternative Atlantic Salmon Investment Market and raised US $30 million 2015 FDA approval for marketing - Fish are now sold in USA market Du, S.J., Z. Gong, G.L. Fletcher, M.A. Shears, M.J. King, D.R. Idler, and C.L. Hew (1992) Growth enhancement in transgenic Atlantic salmon by use of fish antifreeze/growth hormone chimeric gene constructs. Bio/Technology 10:176- 181.  Growth hormone transgenic fish opAFP-sGH chimeric gene construct Ocean pout AFP Salmon Growth Hormone gene Ocean pout AFP Promoter termination signal AFP=antifreeze protein gene Chinook salmon Carp Atlantic Salmon Pacific salmon Tilapia Catfish Rainbow Trout Loach Directed Evolution in Petri Dish Frances Arnold (left), George Smith (middle) and Gregory Winter (right) have won the Nobel Prize for their work bringing evolution into the laboratory to design custom molecules 7 Directed evolution is a powerful technique used in both chemistry and biology to engineer and optimize biomolecules such as enzymes and proteins. It involves mimicking the process of natural evolution in a laboratory setting to create molecules with desired properties or functions. The procedure relies on intended variation of protein sequences at a defined level of randomness. This is coupled to engineered screening and selection strategies. 8  Directed evolution is used to optimize biomolecules in a laboratory setting Create library of slightly a DiFFerent Enzyme. ㉖ Repeat the mimick natural process for Selection. best activity https://www.sciencedirect.com/science/article/abs/pii/S1367593114001240 9 「 小  Each gene contain a random mutation. Directed evolution with comparison to natural evolution The inner cycle indicates the 3 stages of the directed evolution cycle with the natural process being mimicked in brackets https://en.wikipedia.org/wiki/Directed_evolution In 1984, Manfred Eigen published a theoretical paper outlining a possible workflow for directed evolution of enzymes a stepwise iterative 12 Cloning of Green Fluorescence Protein from Aequorea victoria Science (1996). 273 (5280): 1392 Prasher et al., Gene 1992 13 The potential of GFP as a live cell marker… Chalfie et al., Science 1994 14 Improving wild type GFP Roger Tsien 15 Improving wild type GFP Brighter variant S65T (EGFP) brighter Version of much GFP Heim et al., Nature 1995 16 Improving wild type GFP mutation can generate GEPOF DiFFerent Colour. Heim et al., PNAS 1994 17  Improving/changing/creating enzymes using directed evolution She wants the to work in caryme alcshol solvent WT substilisin (enzyme that cuts milk protein casein) Error-prone PCR A library of substilisin variants Activity selection in 35% DMF One substilisin mutant with Frances Arnold increased activity than WT https://www.chemistryworld.com/news/w hoo do you knouthis hat-is-directed-evolution-and-why-did-it- is est? win-the-chemistry-nobel- prize/3009584.article 3x selection --> an evolved substilin with >250 fold increased activity than WT. 18 DNA shuffling- genetic recombination in test tubes DNA Shuffling (Willem Stemmer, 1994) Example shown is at gene level, could imagine this at genome level… Phage display George Smith Gregory Winter 20 George Smith Invented Phage display George Smith used phages 21  George Smith invented phage display specific antibody Fsh For the gene to (dentity the Gene of Interest use a o From a large library From phenotype to genotype ”Out-dated” method since genome projects. 22 that code For Use phage as Vector o FusethegeneofInterestintoPhage's gene its Coat Genewillbedisplayed, n. on the surtace protein of interest 一 Gregory Winter revitalized phage display Antibodies can help to fight diseases. HIgh-specificity and high-affinity Antibodies can be hard to produce using conventional methods. 23  Conventionally , Animals are used to produce Antibody. Antibody production PurnFied Purified Protein of antibody, u 1 Interest r Chemically conjugated Protein oF Interest Beads 24  Antibody production farget different parts of the protein Because there might be different in ant ibodie , , thatan semm bind to Fuse spleen cells cells) the photein wrth cels ( Blood cancer at the vs myeloma But not be site intended. 25 https://www.creative-diagnostics.com/polyclonal-vs-monoclonal-antibodies.htm 一  George Smith uses phage display to evolve antibodies Generation of highly specific and fully Wtein 1 human antibodies in bind vitro. region. Fragment antigen-binding 26 一 George Smith uses phage display to evolve antibodies generate variants of antibody → screen For highest afFinity. 27  First Antibody developed through Directed Evolution HUMIRA® (adalimumab) - A Biologic Medication CPhage Display) One of the notable achievements of Sir Gregory Winter was the development of the first therapeutic antibody, called Humira (adalimumab), which was approved by the U.S. Food and Drug Administration (FDA) in 2002. Humira is used to treat various autoimmune diseases, including rheumatoid arthritis, psoriasis, and Crohn's disease. It has since become one of the best-selling and most widely prescribed drugs worldwide. 28 Directed evolution enhanced by computational tools or DNA shuffling 29 Directed evolution enhanced by computational tools 30  Directed evolution and synthetic biology 31 doi: 10.1039/d2cb00231k Directed evolution lecture by Frances Arnold https://www.youtube.com/watch?v=6hOZ5e0g9Uo 32 Research article for CA1 Thursday 19 Sept, starting at 4:15pm. Open-book, mostly “open-internet”. This CA is an individual human effort. Any communication with others - human or AI – is NOT allowed. NO ChatGPT. 1-hour exam, 30 MCQ and 2 short answer questions. ~Half of the questions are on the research article. ~Half on the lectures. Only main figures (1-5). Supplementary info will not be tested. https://www.science.org/doi/10.1126/science.abg3029 33