Download Poster - iGEM 2006

Cancer Stickybots
Duke University Genetically Engineered Machines 2006
Austen Heinz, Nirav Lakhani, and Lingchong You
Durham, North Carolina 27708, U.S.A.
Regulated Suicide
Nanobody Display
Regulated Suicide Test
1.4
1/1000 4uM IPTG 200 nM 3O6HSL
1/1000 4uM IPTG 200 nM 3O6HSL
1.2
1/1000 4uM IPTG
1/1000 4uM IPTG
1
1/20 4uM IPTG
Abstract
RFP to access circuit functionality.
OD600
1/20 4uM IPTG
0.6
1/1000 4uM IPTG 200 nM 3O6HSL
0.4
0.2
0
0.
17
0.
83
1.
50
2.
17
2.
83
3.
50
4.
17
4.
83
5.
50
6.
17
6.
83
7.
50
8.
17
Quorum sensing sender device.
Quorum sensing receiver device.
Quorum sensing dependent transcription of
anti-toxin protein CcdA.
IPTG activation of CcdB death toxin.
0.8
Hours
Targeted Localization:
Surface expression
of Carcenogenic Embryonic Antigen (CEA) via
auto transporter display enables colon cancer
cell binding specificity.
Discriminate Killing: Invasin allows for
cancer cell invasion while Cytosine Deaminase
allows for dose dependent killing of cancer
cells through the addition of 5-fluorocytosine.
Expression of both genes are linked to quorum
sensing.
Regulated Suicide: Bacteria at low density
(those that do not bind or invade cancer cells)
express the toxin CcdB and commit suicide.
However bacteria in the high density
environment surrounding the tumor express
the antitoxin CcdA via quorum sensing
resulting in their survival.
Targeted Localization
GFP to assess circuit function
PelB leader sequence-directs the protein
to the periplasmic membrane of E.coli
Surface expression of the cAb-CEA5
Nanobodies™ received from Ablynx® +
S*Tag Fusion protein for display detection
C-IgAP Autotransporter surface display
provides a modular scaffold
Discriminate Killing
Suicide at low density via CcdB toxin poisoning
of DNA Gyrase
Density Dependent Growth
1.2
1
0.8
Quorum sensing receiver device
Production of Invasin which allows for
mammalian cell invasion linked to quorum
sensing
Cytosine Deaminase, which converts
nontoxic 5-Fluorocytosine to 5-Fluorouracil
also under control of quorum sensing.
CFP to assess circuit function.
Mammalian Cell Invasion via Invasin
Killing via Cytosine Deaminase conversion of 5-
Fluorocytosine to cancer poisoning 5-Fluorouracil
5FC
5FU
OD6000.6
0.4
Rescue at high population density via CcdA antitoxin
inactivation of CcdB toxin
Modeling
0.2
0
High Cell
Density
S1
Low Cell
Denisty
Low Cell
Density with
AHL
ACKNOWLEDGEMENTS
Jingdong Tian (Duke)
Chris Anderson (UCSF)
Ralph Isberg (Tufts)
Margaret Black (Washington State University)
Serge Muyldermans (Vrije University Brussels)