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Paris iGEM 2007 Doug Tischer Goal • To engineer the first multicellular bacterium to have two distinct cell lines: the soma and the germline. Motivation 1. Limited number of well characterized, frequently used parts. 2. Can engineer in more complexity. 3. Production of toxic compounds. http://www.defra.gov.uk/environment/chemicals/images/toxic.gif The Soma • ftsK- & ΔdapA: Sterile and excretes excess DAP • ftsK: Gene essential for replication ▫ Not in operon ▫ Normal function: thermo sensitive filamentous gene. ▫ Deleted through a controllable recombination event • dapA from B. subtilis is insensitive negative regulation by DAP. Excess DAP is excreted. • Why is DAP excreted…? DapA The Germline DapB • …to feed the germline! • ftsK+ & dapA-: Auxotroph for DAP but can replicate • dapA is an essential gene in the peptidoglycan and lysine biosynthesis pathways DapC DapD DapE DapE • How does this differentiate into the soma? DAP LysA http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=DAPLYSINESYN-PWY Differentiation The cassette: The germline: Cre Recombination The soma: Differentiation Cont. • To maximize growth, have two conflicting constraints: 1. 2. Maximize germline to grow fast Maximize soma to adequately feed germline • Optimum differentiation rate is between 0-50% • Two solutions: 1. 2. Put Cre in pBad so as to control differentiation rate with arabinose. Cre expression under PAD sensitive promotor. Dynamic expression. Assembly • Cloning the ftsK gene proved too difficult. • Assembled cassette in vivo, in a dapA- E. coli strain. (CmR = chloramphenicol resistance) Assembly Cont. Results • Coculturing with prototrophic strain extends dapA- lifespan • Prototrophic cells excrete some DAP (data not shown) ▫ Not enough to sustain dapA-▫ “Spent” media needed less DAP to grow dapA- cells dapA- Strain Survival in LB Coculture dapA- & prototrophic dapA- Results cont: Cre recombination rate 1. lox-KmR-lox: Kanamycin screening ▫ No observable colonies 2. lox-KmR-lox: Growth in Kanamycin ▫ 36.8% Cre recombination rate 3. lox-gfp-T-lox-mrfp ▫ (Yet to be done) Were they successful? • Goal: To engineer the first multicellular bacterium to have two distinct cell lines - the soma and the germline. Interesting and Exciting • Monitor changes in soma/germ genome & phenotype ▫ Do they swap genes? ▫ Do they become more or less dependent? • Directed evolution of soma ▫ More possibilities because doesn’t have to reproduce ▫ Optimized production of cytotoxic compounds • Biological Security ▫ Induce full conversion of germsoma ▫ Will not persist in environment ▫ Bioremiation http://www.thecrcenter.com/wpcontent/uploads/2007/06/Hazmat%20Incident.pn g References • http://parts.mit.edu/igem07/index.php/Paris ▫ All images are from this site unless otherwise noted • http://biocyc.org/ECOLI/NEWIMAGE?type=PATHWAY&object=DAPLYSINES YN-PWY