Transcript X-Verter - iGEM 2006

Duke iGEM 2006
Duke University
international Genetically Engineered Machines Jamboree 2006
M.I.T., Cambridge MA, 02139 U.S.A.
November 4-5, 2006
staff
Thom LaBean
Faisal Reza
Jingdong Tian
Lingchong You
Fan Yuan
students
Keddy Chandran
Hattie Chung
Matt Feltz
Austen Heinz
Sagar Indurkhya
Eric Josephs
Nirav Lakhani
John Lee
Steven Lin
Pat O’Brien
Nicholas Tang
Bryan Van Dyke
2006 Projects
Engineering Synthetic Oscillatory Gene
Networks at the Population Level
Sagar Indurkhya
Nicholas Tang
Austen Heinz
Lingchong You
Computational Chemistry
X-Verter
X-Verter
Expression by Small Molecule Promotion
d tetR 
dt
d  cinI RBS 
dt
d  cinR 
dt
d CFP 
dt
 HSLRhl 
= Vmax, rhl
K m, rhl +  HSLRhl 
= Vmax, rhl
K m, rhl +  HSLRhl 
= Vmax, rhl
K m, rhl +  HSLRhl 
= Vmax, rhl
K m, rhl +  HSLRhl 
d lacI 
 HSLRhl 
K m, cin +  HSLCin 
= Vmax, cin
K m, cin +  HSLCin 
= Vmax, cin
K m, cin +  HSLCin 
= Vmax, cin
K m, cin +  HSLCin 
d luxI RBS 
dt
 HSLRhl 
d luxR 
dt
 HSLRhl 
d YFP 
dt
 HSLCin 
= Vmax, cin
dt
d  cI l 
= Vmax, lux
dt
 HSLCin 
d  rhlI RBS 
d  cinR 
dt
 HSLCin 
d CFP 
dt
 HSLLux 
= Vmax, lux
K m, lux +  HSLLux 
= Vmax, lux
K m, lux +  HSLLux 
= Vmax, lux
K m, lux +  HSLLux 
dt
 HSLCin 
 HSLLux 
K m, lux +  HSLLux 
 HSLLux 
 HSLLux 
Expression by Gene Repression
d  rhlI mRNA 
dt
=
H tet K m, tet
K m, tet + tetR 
d  cinI mRNA 
dt
=
H lac K m, lac
K m, lac + lacI 
d luxI mRNA 
dt
=
H cI l K m, cI l
K m, cI l + cI l 
Formation of HSL Molecule
d  HSLCin 
dt
d  cinI 
dt
d  HSLLux 
= k HSL Formation  cinR  cinI 
dt
d luxI 
= k Protein Formation  cinRBS  cinmRNA 
dt
d  HSLRhl 
= k HSL Formation luxR luxI 
= k Protein Formation luxRBS luxmRNA 
dt
d  rhlI 
dt
= k HSL Formation  rhlR  rhlI 
= k Protein Formation  rhlRBS  rhlmRNA 
Degradation of Molecules
d  RFP 
dt
d CFP 
dt
d YFP 
dt
d  HSLlux 
dt
d  cinI RBS 
dt
d  cinI mRNA 
dt
= k LVA. Protein Deg.  RFP 
= k LVA. Protein Deg. CFP 
= k LVA. Protein Deg. YFP 
= k HSL Deg.  HSLlux 
= k Protein Deg.  cinI RBS 
= k Protein Deg.  cinI RBS 
d  cinI 
dt
d luxI 
= k Protein Deg.  cinI 
dt
d  rhlI 
dt
d  HSLcin 
= k Protein Deg. luxI 
= k Protein Deg.  rhlI 
dt
d  rhlI RBS 
dt
d  rhlI mRNA 
dt
= k HSL Deg.  HSLcin 
= k Protein Deg.  rhlI RBS 
= k Protein Deg.  rhlI mRNA 
d luxR 
dt
d luxR 
dt
d  rhlR 
dt
d  HSLrhl 
dt
d luxI RBS 
= k Protein Deg.  cinR 
= k Protein Deg. luxR 
= k Protein Deg.  rhlR 
= k HSL Deg.  HSLrhl 
dt
d luxI mRNA 
dt
= k Protein Deg. luxI RBS 
= k Protein Deg. luxI mRNA 
X-Verter Modeling Results
Predator-Prey
Expression by Small Molecule Promotion
d  BlipPrey 
 HSLCin 
= Vmax, cin
dt
K m, cin +  HSLCin 
d  AmpRPredator 
dt
= Vmax, lux
 HSLLux 
K m, lux +  HSLLux 
Formation of HSL Molecule
d  HSLCin 
d  HSLLux 
= k HSL Formation  cinR  cinI 
dt
Degradation of Molecules
d GFP 
dt
d  cinI 
= k LVA. Protein Deg. GFP 
dt
d  RFP 
dt
d luxI 
= k LVA. Protein Deg.  RFP 
dt
d  AmpR 
dt
d  HSLlux 
= k Protein Deg.  AmpR 
dt
d  BlipPredator 
= k Protein Deg.  BlipPredator 
dt
Expression by Gene Repression
d  RFP 
dt
d GFP 
dt
=
H lac K lac
K lac + lacI 
=
H lac K lac
K lac + lacI 
d  BlipPredator 
dt
=
d luxR 
H lac K lac
K lac + lacI 
dt
d luxI 
dt
= k HSL Formation luxR  luxI 
= k Protein Deg.  cinI 
= k Protein Deg. luxI 
= k HSL Deg.  HSLlux 
dt
d  BlipPrey 
= k Protein Deg.  BlipPrey 
dt
=
H lac K lac
K lac + tetR 
=
H lac K lac
K lac + lacI 
d  cinR 
dt
d  cinI 
dt
d luxR 
dt
d luxR 
dt
d  HSLcin 
dt
=
H lac K lac
K lac + lacI 
=
H lac K lac
K lac + tetR 
= k Protein Deg.  cinR 
= k Protein Deg. luxR 
= k HSL Deg.  HSLcin 
Predator-Prey Modeling
Biobricks Manager
Conclusion
Computational Chemistry
Derived pH degradation rates
X-Verter (3-Stage Synchronized Oscillator)
Designed and Modeled
Predator -Prey (2-Stage Synchronized Oscillator)
Designed and Modeled
Nearly Completed with Assembly
Biobricks Manager (Biological Circuit IDE)
Soon to be released as open-source
Experimental Characterization
Experimental Characterization
Obtained specificity and latency graphs for characterization
Encoding Information In Vivo with DNA and Light
Austen Heinz
Keddy Chandran
Pat O’Brien
Fan Yuan
Human Encryption System
Creating a DNA Alphabet
Symbol
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Code
AAA
AAC
AAG
AAT
ACA
ACC
ACG
ACT
AGA
AGC
AGG
AGT
ATA
ATC
ATT
CAA
Symbol
Q
R
S
T
U
V
W
X
Y
Z
1
2
3
4
5
6
Code
CAC
CAG
CCA
CCC
CCG
CCT
CGA
CGC
CGG
CGT
CTA
CTC
CTG
CTT
GAA
GAC
Symbol
7
8
9
0
+
*
/
=
>=
<=
!
?
.
"
Code
GAG
GAT
GCA
GCC
GCG
GCT
GGA
GGC
GGG
GGT
GTA
GTC
GTG
GTT
TAA
TAC
Symbol
;
,
(
)
[
]
<
>
@
#
^
&
%
Code
TAG
TAT
TCA
TCC
TCG
TCT
TGA
TGC
TGG
TGT
TTA
TTC
TTG
TTT
UNUSED CAT
UNUSED ATG
Creating a Light Alphabet
Red-Shifting
Step 1
LuxC
XbaI Removal
LuxD
LuxA
LuxB
LuxE
C106V
A75G
Step 2
LuxA
C106V
A75G
Step 3
LuxA
V173A
LuxA
A75G
V173A
LuxA
C106V
A75G V173A
Step 4
LuxA
LuxA
C106V
V173A
LuxA
Creating a Light Alphabet
Wavelength Scanning
PSB417 Intensity (a.u.)
600
500
Intensity
400
300
PSB417 Intensity (a.u.)
200
100
0
0
100
200
300
400
-100
Wavelength
500
600
700
Creating a Light Alphabet
Peak Wavelength
In Vivo Imaging
T=0
T=17 Hours
Applications
National Security
Applications
Health
Conclusions
Mission Accomplished
A Novel Suicide Circuit for Tumor
Targeting Bacteria
Austen Heinz
Nirav Lakhani
Lingchong You
Micromachines and Swarm Behavior
Stickybots
Targeted Localization
A Sticky Swarm
Targeted Localization
Circuit Function:
GFP to assess circuit operation
Constitutive Expression of:
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
Targeted Localization
Nanobodies
Neisseria gonorrhea IgA Protease
Discriminate Killing
Controllable Killers
Discriminate Killing
Key Characteristics:
Quorum sensing receiver device
Production of Invasin linked to population density
Production of Cytosine Deaminase, which converts
nontoxic 5-Fluorocytosine to 5-Fluorouracil also under
control of quorum sensing.
CFP.
Discriminate Killing
Mammalian Cell Invasion via Invasin
Killing via Cytosine Deaminase conversion of 5-
Fluorocytosine to cancer poisoning 5-Fluorouracil
5FC
5FU
Regulated Suicide
Stickybot Self Destruction
Regulated Suicide
Key Characteristics:
Quorum sensing receiver device.
Quorum sensing dependent transcription of antitoxin protein CcdA
IPTG activation of CcdB death toxin.
RFP
Regulated Suicide
CcdB/GyrA59
CcdA/CcdB
Regulated Suicide
Regulated Suicide
1.4
1.2
OD600
1
1/1000 4uM IPTG 200 nM 3O6HSL
1/1000 4uM IPTG 200 nM 3O6HSL
1/1000 4uM IPTG
1/1000 4uM IPTG
1/20 4uM IPTG
0.8
1/20 4uM IPTG
0.6
0.4
0.2
0
17 .83 .50 .17 .83 .50 .17 .83 .50 .17 .83 .50 .17
0
1
2
2
3
4
4
5
6
6
7
8
0.
Time(h)
Regulated Suicide
1.2
1
0.8
0.6
OD600
0.4
0.2
0
High Cell
Density
S1
Low Cell
Denisty
Low Cell
Density with
AHL
Results
Conclusion
Working Regulated Suicide Circuit and
System Modeling
Mission Accomplished
Engineering “Sticky” Magnetic Bacteria for
Power Generation
Eric Josephs
Hattie Chung
Jingdong Tian
Thom LaBean
Bacterial Dynamo
What’s a dynamo?
This is a dynamo:
But if we want to make
this out of bacteria,
where are we going to
find magnets? Looks
like we’re going to
have to ask our good
friend...
http://www.houseofcuss.com/hocvault/thepipe/archives/2005_09.shtml
Bacterial Dynamo
MAGNETOSPIRILLUM SP. AMB-1!
http://magnum.mpi-bremen.de/magneto/research/index.html
Spinning Tethered Bacteria
QuickTime™ and a
MPEG-4 Video decompressor
are needed to see this picture.
Bacterial Dynamo
Bacterial flagellar proteins are easy to modify.
If a flagella sticks to a surface, it will cause the cell body to spin.
Some bacteria grow chains of nano-sized magnets in their cell bodies.
A spinning magnet field will induce a voltage in a coil.
If we engineer the flagella of magnetic bacteria to
stick to a surface above a coil, we can get a dynamo
powered by flagellar motors.
This concept has been proven before by sticking magnetic bacteria to
coils with anti-flagellin antibodies and the system fell apart after a few
hours (days?). If we genetically engineer the bacteria to produce a
flagellin protein that sticks to an easily patterned surface, the system
will ‘self-assemble’ and could continue indefinitely.
Bacterial Dynamo
Making ‘sticky’ flagella
Find protein which binds to
hard-baked S-1813 positive
photoresist by screen
~10^8 12-aa random
peptides expre ssed on
flagellar exterior to see
which bind to photoresist.
Cut out amb0684, AMB-1
flagellin gene, split it in
two, splice in the sticky
gene with thioredoxin
structure.
Put it back into AMB-1, get
STICKY MAGNETIC
BACTERIA.
Making the dynamo
Fabricate a little coil for
magnetic bacteria to
grow on, seal it with
positive photoresist so
the bacteria will stick.
More details at poster.
Coil
Conclusions
Our coil is completed, we isolated a ‘sticky’
peptide, and are currently working on
PCRing out the AMB-1 flagellin gene.
Possible applications of this project include
‘natural’ batteries and, since research is
being conducted in using bacteria to convert
the chemical energy of many different
sources (contaminants, pollution, nuclear
waste) into energy the bacteria can use,
this dynamo could possibly be engineered to
convert almost anything as fuel
Duke iGEM 2006 Conclusions
Testing and characterization of multiple bacteria small molecule
communication systems. Modeling and construction of two synthetic
artificial ecosystems in bacteria: X-Verter a three stage population level
oscillator and Predator Prey a two stage two population oscillator.
Created an open source gene circuit IDE called Biobrick Manager.
Creation and characterization in a mammalian system of a DNA and light
“alphabet” for Human Encryption. Future uses include national security
and health detection applications
Development of a working bacteria circuit that causes the bacteria to selfdestruct when outside the cancer environment for the Cancer Stickybots
project and system modeling.
Evolving E coli bacteria to stick to positive photoresist and
micromachining of an apparatus for future use as a Bacterial Dynamo:
magnetic bacteria that spin above a coil and produce electricity via
Faraday’s law.
Acknowlegements
Jingdong Tian (Duke)
Fan Yuan (Duke)
Thom LaBean (Duke)
Lingchong You (Duke)
Faisal Reza (Duke)
Myra Halpin (NCSSM)
Bob Gotwals (NCSSM)
Ralph Isberg (Tufts)
Chris Anderson (UCSF)
Margaret Black (Washington State)
Serge Muyldermans (VIB)
Chandra Drennen (USC)
Andrew Simnick (Duke)
Ashutosh Chilkoti (Duke)
Mike Winson (University of Wales Aberystwth)
Eric Metcalf (University of Illinois)