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Paris iGEM 2007

Paris iGEM 2007. Doug Tischer. Goal. To engineer the first multicellular bacterium to have two distinct cell lines: the soma and the germline. Motivation. Limited number of well characterized, frequently used parts. Can engineer in more complexity. Production of toxic compounds.

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Paris iGEM 2007

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  1. ParisiGEM 2007 Doug Tischer

  2. Goal • To engineer the first multicellular bacterium to have two distinct cell lines: the soma and the germline.

  3. Motivation • Limited number of well characterized, frequently used parts. • Can engineer in more complexity. • Production of toxic compounds. http://www.defra.gov.uk/environment/chemicals/images/toxic.gif

  4. 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…?

  5. The Germline DapA 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 • How does this differentiate into the soma? DapC DapD DapE DapE DAP LysA http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=DAPLYSINESYN-PWY

  6. Differentiation The cassette: The germline: Cre Recombination The soma:

  7. Differentiation Cont. • To maximize growth, have two conflicting constraints: • Maximize germline to grow fast • Maximize soma to adequately feed germline • Optimum differentiation rate is between 0-50% • Two solutions: • Put Cre in pBad so as to control differentiation rate with arabinose. • Cre expression under PAD sensitive promotor. Dynamic expression.

  8. Assembly • Cloning the ftsK gene proved too difficult. • Assembled cassette in vivo, in a dapA- E. coli strain. (CmR = chloramphenicol resistance)

  9. Assembly Cont.

  10. Results dapA- Strain Survival in LB • 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 Coculture dapA- & prototrophic dapA-

  11. Results cont: Cre recombination rate • lox-KmR-lox: Kanamycin screening • No observable colonies • lox-KmR-lox: Growth in Kanamycin • 36.8% Cre recombination rate • lox-gfp-T-lox-mrfp • (Yet to be done)

  12. Were they successful? • Goal: To engineer the first multicellular bacterium to have two distinct cell lines - the soma and the germline.

  13. 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 germsoma • Will not persist in environment • Bioremiation http://www.thecrcenter.com/wp-content/uploads/2007/06/Hazmat%20Incident.png

  14. References • http://parts.mit.edu/igem07/index.php/Paris • All images are from this site unless otherwise noted • http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=DAPLYSINESYN-PWY

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