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Yale iGEM SYNTHETIC BIOLOGY: IMPLEMENTATION AND IMPACT

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Modification of existing organisms What is Synthetic Biology?

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with the goal of applying these novel organisms for the betterment of mankind. What is Synthetic Biology?

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A brief history of synthetic biology Case Study: artemisinin and synthetic biology Biofuels and synthetic biology Agriculture and synthetic biology Obstacles, ethical issues, and looking to the future Yales iGEM project So whats the game plan for today?

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Watson and Crick discovered the structure of the DNA molecule with the invaluable help of Rosalind Franklin 1953 Enter James Watson, Francis Crick, and Rosalind Franklin

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Models and Photographs Structure of DNA

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Each DNA molecule is composed of two strands which are made of a phosphate backbone and are connected in the middle by nucleotides Four nucleotides: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T) Key point: each one binds to only one other: A to T and C to G One pair of A-T or C-G is called a Base Pair Structure of DNA

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Allows DNA to replicate and create identical new strands Demonstrated how information is stored and passed along in cells Importance of the structure of DNA

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A gene is simply a sequence of nucleotide base pairs, something like AAGGATCCACTGAGATTACCA, which codes for a protein. What are genes?

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DNA RNA Protein How do genes code proteins?

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Cells dont just continually produce proteins their genes are always regulated and controlled by a series of genetic parts, such as switches, promoters, and repressors. Genes are constantly regulated

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The system works by using a protein called a repressor When there is no lactose present, the repressor binds to the gene which codes for lactose-digesting proteins. This prevents the proteins from being created When lactose is introduced to the cell, the repressor reacts with the lactose and detaches from the DNA. As a result, lactose-digesting proteins are created again Lac operon system

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Thus, proteins are only made when they are needed, i.e. when lactose is present!

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Well, so what? Why should we care?

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CASE STUDY: ARTEMISININ AND SYNTHETIC BIOLOGY

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In 2012, malaria caused between 473,000 and 789,000 deaths worldwide, primarily in sub- Saharan Africa Malaria accounts for one in every five childrens deaths globally The Plague of Malaria

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Currently, the most popular treatment for Malaria is Quinine Excessive usage after World War II led to the Malaria parasite developing a resistance to the drug. Resistance is extremely dangerous - people will not be able to be cured if the medicine doesnt work Current treatment, quinine, and its failure

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Recently discovered potent antimalarial drug Artemisinin can be used to its full potency to fight malaria, unlike the now almost-defunct Quinine The advent of artemisinin

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Artemisinin is derived from the Sweet Wormwood plant Cultivation is a hindrance to its cheap and easy global distribution But distribution MUST be cheap and easy in impoverished countries! However

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The supply of artemisinin is influenced heavily by farming conditions Leads to fluctuations in supply and prices Not viable as Quinine replacement However pt. 2

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A way to create cheap synthetic artemisinin was pioneered by Stanford professor Jay Keasling Synthetic Biology Comes into Play

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A simple organism was chosen to manufacture this artemisinin: Yeast Synthetic genetic circuit + natural DNA reading/transcribing mechanism = yeast cell which produces artemisinin Cells do the work

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Hundreds of thousands of farmers in developing countries rely on sweet wormwood farming Synthetic artemisinin would eliminate need for sweet wormwood cultivation Puts hundreds of thousands of farmers out of a job Economic Impact of Synthetic Artemisinin

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Recall this graph: Introduction of cheap synthetic-made artemisinin would stabilize prices (and lower them) More accessible to those at risk for malaria Economic Impact of Synthetic Artemisinin

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Which is more important? Stable prices and accessibility to medicine for millions at risk for malaria OR The livelihoods of hundreds of thousands of farmers Is it worth it?

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BIOFUELS AND SYNTHETIC BIOLOGY

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Most fuels used currently are extracted from the earth Unsustainable and cause pollution Fossil Fuels

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A fuel which is synthesized from organic resources Scientists are developing engineered cells which consume biomass and produce fuels An alternative: biofuels

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Already naturally made in small quantities by cells via anaerobic respiration For starters, Ethanol

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The amount of ethanol secreted by bacteria naturally is nowhere near enough to power a car! The problem

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Insertion of a gene which increases the amount of ATP and NADH (energy-providing molecules) in a cell. More energy, more ethanol naturally produced Knocking out all chemical processes except the fermentation of ethanol using chemical means A few solutions

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There is one organism on which companies are focusing their time, effort, and hundreds of millions of dollars: algae The yeast of biofuels

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Single Functionality Advantages of using algae as a biofuel producer Uses of Corn Uses of Algae Alcohol Livestock feed Human feed Cooking oil Bakery Products Soap Insecticides Ethanol Ceramics Adhesives Flour Green Stuff in Fancy Drinks Ethanol Per-acre production: Algae can produce between 9,000 and 61,000 liters of fuel per hectare per year, compared to corns maximum of 5,000 liters Infinite energy - photosynthesizing

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Example: land required to displace all gasoline in the united states, by method

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Algae live in water large water tanks required Maintenance, storage, etc Expensive Processing Transport Current obstacles

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Insertion of a gene which makes algae communal they dont hoard sunlight from other cells. Sharing is caring! In-Progress Solutions

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Insertion of a human gene into algae: The gene that codes carbonic anhydrase (CA)II CAII regulates CO2 levels in human red blood cells by combining CO2 and H20 into bicarbonate and protons In algae, CAII converts excess C atoms into CO2, which is used during photosynthesis to produce energy More naturally-produced energy = more energy for production of biofuels = cheaper fuels = commercial viability Get Humans involved

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In the United States alone, there are 1.3 Billion tons of unused biomass Biomass is used to feed microbes which manufacture biofuels; With this much biomass, we have the potential to replace the domestic production of oil Episode IV: A New Hope

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OBSTACLES AND LOOKING TO THE FUTURE

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How to asses emerging tech Public Beneficence Is the technology helpful for people? Responsible Stewardship Humans responsible for human safety and for earths safety Intellectual freedom and responsibility Pursuing science safely and without harm to others Democratic deliberation Civil, public exchange of opinions and ideas Justice and fairness Commitment to all groups sharing benefits and burdens equally Ethical Considerations

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Public beneficence How will synthetic biology benefit humanity? Medicine Biofuels Energy Agriculture Responsible Stewardship Ethics education Collaboration between government and scientists Applying these considerations to Synthetic Biology

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Intellectual freedom and responsibility Garage Engineering Uncontrolled Release Bioterrorism Democratic Deliberation Improving scientific literacy Validating scientific claims about genetic engineering Justice and Fairness Managing risks of pathogens Applications of Synthetic Biology benefiting all Applying these considerations to Synthetic Biology

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PORTING MULTIPLEX AUTOMATED GENOME ENGINEERING INTO OTHER EUBACTERIA Yale iGEM 2015

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MULTIPLEX AUTOMATED GENOME ENGINEERING (MAGE)

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MAGE PROCESS Subject matter experts: George Church, Ph.D., Frederic Vigneault, Ph.D., M.Sc. Producer/writer: Rick Groleau Graphic design and technical development: Lenni Armstrong, Jamie Ciocco

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RHIZOBIA ARE NITROGEN-FIXING BACTERIA Rhizobia fix nitrogen in root nodules of legumes Symbiotic relationship Rhizobia fix Nitrogen Legumes provide essential molecules

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NITROGEN-POOR SOIL: AN ISSUE IN AGRICULTURE Relative yield losses in corn crops in Sub-Saharan Africa. Fischer et al. 2014. Punchstock

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Cyanobacteria Photosynthetic aquatic bacteria Can grow off of light and trace metals net gain in energy production when harvesting CYANOBACTERIA: PHOTOSYNTHETIC BACTERIA

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Challenges to Overcome Finding beta- homologues Transforming plasmid into cells Selecting for transformants MutS Knockout PORTING MAGE INTO OTHER EUBACTERIA

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IDENTIFYING BETA HOMOLOGS Lambda-red beta protein is specific to E. Coli Tools for comparing nucleotide sequences NCBI Blast HMMER

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VECTOR DESIGN FOR EXPRESSION OF RECOMBINASE Broad-host range plasmid pKT230

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TRANSFORMATION AND SELECTION ELECTROPORATION Kan R

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PROOF OF CONCEPT: CITRINE AS A SCREEN FOR MAGE Wang and Isaacs et. al 2009

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THANK YOU, EVERYONE!

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Want to know more about iGEM? (http://igem.org/About) Want to start a team? http://2015.igem.org/Starting_a_Team MORE ABOUT IGEM