Bo Keun Song, Ee Seul Shin, Yu-Lim Lee and Jae Sung YooAdvisor: Parisutham Vinuselvi and Sung Kuk Lee, Ph.D.

Synthetic Biology Lab, Ulsan National Institute of Science and Technology, Korea

AbstractSynthetic microbes are rapidly gaining use as a host in biofuel production, bioremediation, pharmaceutical and many other biorefinery applications.Despite these wide and far-reaching influences, however, synthetic microbes do not experience a warm welcome from the general public becauseof the potential hazards that might endanger our biosphere. Although there have been proposals for killing synthetic microbes after accomplishingtheir missions, none of those devices could eliminate the adverse effects of engineered DNA. Here we, UNIST_KOREA team, propose and develop anovel device that makes synthetic microbes efficiently perform programmed cell death and DNA degradation when exposed to naturalenvironment.

Chop coli ModulesSensor, Processor and Lysis modules constitute ChoCoLate device.

ChoCoLate device helps Escherichia coli to distinguish between natural environment and fermentor based on cues such as light, temperatureand quorum sensing chemicals. Based on this high-fidelity sensing, we try to construct a high-performance DNA lysis device using tightlyregulatable processor modules.

Processor ModulesSensor Modules Lysis Modules

1. Optical Sensor

Fermentor – Dark;

Natural environment - Light.

Chop. coli senses its environment

using three distinct kinds of sensors:

Optical, Physical and Chemical sensor.

2. Physical Sensor

Tfermentor = 37 ⁰C,

Tenvironment = 4 - 30 ⁰C.

.

3. Chemical Sensor

Fermentor has uniform quorum-sensing

molecule AI-2.

Environment has a mixture of quorum

sensing molecules like AIP and AHL.

Processor module helps reduce the leaky level and avoid undesired cell death.

1. fim Inversion System

The fim inversion system

would help reduce the leaky

level and favor a tight

regulation of gene expression.

GFP

IRR IRL

OFF

GFP

IRR IRL

ON

2. CI Control System

CI control system would

help reduce the undesired

cell death by delaying the

time needed to activate

gene expression.

PompC

RBS cI

RBScIbinding

siteGOI

PL

Physical SensorOptical Sensor

CI ProcessorApplication

Osmo- Regulatory Sensor

FimE

Lysis module would help eradicatethe GMOs safely from the environment.

Chop. coli prefers darkness over light.

Darkness represses the lytic device.

Crosstalk between light and osmo-receptor makes

Chop. coli sense osmolality.

High sugar concentration represses the lytic device.

Chop. coli prefers high temperature.

High temperature represses the lytic device.

0

10000

20000

30000

40000

50000

60000

0 2 4 6 8 10

Early- Log Phase

Mid Log Phase

Late Log Phase

Control

Cultivation Time (Hours)

Rel

ati

ve

Flu

ore

scen

ce U

nit

The CI repressor helps delay the duration of lysis by

one hour after the signal is being received. This would

help prevent undesired lysis when processing the Chop

coli on lab bench.

Future Work Eradicates the fear on GMOs.

Ensures no horizontal gene transfer.

Helps in the formation of proto-cells useful for

synthetic genome transplantation (Nature 2011).

AcknowledgementsWe would like to thank Profs. Cheol-Min Ghim and Yoon-Kyoung Cho andmembers of Synthetic Biology Laboratory (SKLee lab) for their constructivecriticism and helpful suggestions. We appreciate the support from School ofNano-Bioscience and Chemical Engineering at Ulsan National Institute ofScience and Technology (UNIST).

1. Cell Lysis Module

T4 Holin- Endolysin system would

help damage the cell membrane

and cause lysis.

2. DNA Degradation Module

Dpn enzymes would help chop up the DNA

Dpn I- if the host DNA is methylated

Dpn II- if the host DNA is not methylated

Integration of light and quorum sensing system with

fimE

PhyB-PIF3

for enhanced efficiency of DNA lysis of targeted

genetically modified organisms (GMOs).

References1. A. Levskaya et al., Nature 438, 441 (2005).

2. T. S. Ham, S. K. Lee, J. D. Keasling, A. P. Arkin, Biotechnology

and Bioengineering 94, 1 (2006).

3. O. Oleksiuk et al., Cell 145, 312 (2011)

4. J. W. Szostak, D. P. Bartel, P. L. Luisi, Nature 409, 387 (2001)

5. iGEM 2008 UC-Berkeley team for the proposal of Lysis device

Nature (2005)

Cell (2011)

B & B (2006)iGEM 2008 (UC-Berkely)