Expression of dsRNA in Spirogyna algae to inhibit development of Malaria carrying mosquitoesMalariaMalariaAnopheles mosquitoes: A deadly vectorMalaria: Current TreatmentsMalaria: Prevention and Vector Control Research Problem & GoalsResearch OutlineTest Mortality of Larvae Fed on Transgenic AlgaeOutcomesNeeded ResourcesStudy ImplicationsQuestions?ReferencesKAT PAK AND MATTHEW LOPER20.109 FINAL PRESENTATIONDECEMBER 7, 2007Expression of dsRNA in Spirogynaalgae to inhibit development of Malaria carrying mosquitoesCite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Malariay Caused by Plasmodium protozoan parasites{ Once a host is infected, the parasite remains indefinitely y Infects 515 mil people a year and kills between 1 and 3 million including South America, Africa, India, Asia, and the Middle EastMap from Wikipedia, using 2003 data from U.S. CDC.Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Malariay Associated with poverty and possible huge hindrance to economiesy Estimated $12B malaria economic costs in Africa per year1. Sachs J, Malaney P (2002). "The economic and social burden of malaria". Nature 415: 680-5. Image courtesy of “dosh dosh.”Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Anopheles mosquitoes: A deadly vectory Female Anopheles mosquitoes primary Plasmodium carriery Larvae hatch and feed on Spirogyna algae2y Grow and develop tointo pupa and become adults2. Rejmankova, E. H. et al. 1993 Environmental and regional determinants of Anopheles (Diptera: Culicidae) larval distribution in Belize, Central America. Environ. Entomol. 22, 978–992.Courtesy of US CDC.Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Malaria: Current Treatmentsy No effective, approved vaccine{ Huge amount of research being done to discover effective vaccines{ Current drugs not affordable or practical for chronic use in malaria regions{ Recently, a group at Cal used Synthetic Biology to developed yeast to produce artemisinin2 much cheaper than current synthesis methods2. Keasling, Jay D et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature [Nature]. Vol. 440, no. 7086, pp. 940-943. 14 Apr 2006. Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Malaria: Prevention and Vector Control y Widespread use of insecticidesy Domestic spraying of fungus Beauveria bassiana3y Drainage and filling of ponds/rivers{ Huge economic and environmental costs4y Clearing of algae from ponds5y Biological (predator and microbe)larvicidal controls3. Simon Blanford, et al. Fungal Pathogen Reduces Potential for Malaria Transmission. (10 June 2005) Science 308 (5728), 1638. 4. Collins, F. H. & Paskewitz, S. M. 1995. Malaria: current and future prospects for control. A. Rev. Entomol. 40, 195–219.5. WALKER, K. (2007) Contributions of Anopheles larval control to malaria suppression in tropical Africa: review of achievements and potential. Medical and Veterinary Entomology 21(1)Photo removed due to copyright restrictions.People working in a river.Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Research Problem & GoalsGoal:Reduce the Anopheles mosquito population by targeting key larvae genes with dsRNA expressed in transgenic algaeBenefits:y Minimize harm to ecosystemy Directly-targeted approachy Reduce maintenancePhoto removed due to copyright restrictions.Person spraying for mosquitos.Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Cite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Research Outline1) Determine larvae key developmental genes2) Create transgenic algae expressing dsRNA3) Test if larvae fed on transgenic algae have increased mortality 1. artificial diet assays2. transgenic algae 3. growth assaysCite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].1) Construct cDNA library of A. pseudopunctipennislarvaeLarvaDNA2) Prepare dsRNAsdsRNA3) Apply varied doses of dsRNAto larvae diet4) Confirm triggering of RNAi responseDetermine Key Developmental Genes of LarvaeNorthern BlotArtificial diet assayCite as: Angela Belcher, Drew Endy, Natalie Kuldell and Agi Stachowiak. Course materials for 20.109 Laboratory Fundamentals in BiologicalEngineering, Fall 2007. MIT OpenCourseWare (http://ocw.mit.edu), Massachusetts Institute of Technology. Downloaded on [DD Month YYYY].Test Mortality of Larvae Fed on Transgenic AlgaeStrongPromoterdsRNAselectable markerAlgae1. Assemble transformation expression cassette2. Ensure no off-target effect on algae3. Perform growth chamber assaysmicroarray growth chamberOutcomesIf all goes to