ParisiGEM 2007Doug 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.

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…?

The Germline•…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?

http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=DAPLYSINESYN-PWY

DapA

DapB

DapC

DapD

DapE

DapE

LysA

DAP

Differentiation

The cassette:

The germline:

The soma:

CreRecombination

Differentiation Cont.• To maximize growth, have

two conflicting constraints:1. Maximize germline to

grow fast2. Maximize soma to

adequately feed germline• Optimum differentiation

rate is between 0-50%• Two solutions:

1. Put Cre in pBad so as to control differentiation rate with arabinose.

2. 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

CoculturedapA- & prototrophic

dapA-

dapA- Strain Survival in LB

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

References

http://parts.mit.edu/igem07/index.php/Paris