Expression of dsRNA in Spirogyna algae to inhibit development of Malaria carrying mosquitoes KAT PAK AND MATTHEW LOPER 20 109 FINAL PRESENTATION DECEMBER 7 2007 Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Malaria y 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 East Map from Wikiped a using 2003 data from U S CDC i Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Malaria y Associated with poverty and possible huge hindrance to economies y Estimated 12B malaria economic costs in Africa per year 1 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 Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Anopheles mosquitoes A deadly vector y Female Anopheles mosquitoes primary Plasmodium carrier y Larvae hatch and feed on Spirogyna algae2 y Grow and develop to into pupa and become adults Courtesy of US CDC 2 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 Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Malaria Current Treatments y 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 methods 2 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 Biological Engineering 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 insecticides y Domestic spraying of fungus Beauveria bassiana3 y Drainage and filling of ponds rivers Huge economic and environmental costs4 y Clearing of algae from ponds5 y Biological predator and microbe larvicidal controls Photo removed due to copyright restrictions People working in a river 3 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 Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Research Problem Goals Goal Reduce the Anopheles mosquito population by targeting key larvae genes with dsRNA expressed in transgenic algae Benefits y Minimize harm to ecosystem y Directly targeted approach y Reduce maintenance Photo 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 Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Research Outline Determine larvae key developmental genes 2 Create transgenic algae expressing dsRNA 3 Test if larvae fed on transgenic algae have increased mortality 1 1 artificial diet assays 2 transgenic algae 3 growth assays Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Determine Key Developmental Genes of Larvae Larva 1 Construct cDNA library of A pseudopunctipennis DNA larvae 2 Prepare dsRNAs dsRNA 3 Apply varied doses of dsRNA to larvae diet 4 Confirm triggering of RNAi response Northern Blot Artificial diet assay Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Test Mortality of Larvae Fed on Transgenic Algae Strong Promoter dsRNA Algae selectable marker 1 Assemble transformation expression cassette 3 Perform growth chamber assays 2 Ensure no off target effect on algae microarray growth chamber Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw mit edu Massachusetts Institute of Technology Downloaded on DD Month YYYY Outcomes If all goes to plan y Test compatibility of transgenic algae with environment y Introduce transgenic algae to natural habitat y Test prevalence of transgenic algae over time after introduction y Determine if technique can be applied to different food sources mosquito species and locations If nothing goes to plan y Test other dsRNA sequences y Target a food source of the mosquito Cite as Angela Belcher Drew Endy Natalie Kuldell and Agi Stachowiak Course materials for 20 109 Laboratory Fundamentals in Biological Engineering Fall 2007 MIT OpenCourseWare http ocw