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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 germsoma
▫ 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