Transcript Poster - iGEM 2006

Human Encryption
Duke University Genetically Engineered Machines 2006
Austen Heinz, Pat O’Brien, Keddy Chandran, Andrew Simnick, and Fan Yuan
Durham, North Carolina 27708, USA.
Our Coding Schemes
Objective
Symbol
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Conceived as a new means for information storage
and transmission, the Human Encryption project is
a proof-of-concept work that demonstrates that a
symbiotic bacteria containing a modified
luminescent operon, can be used in a mammal
system to function as a detectable marker for a
message stored in the form of DNA.
LuxC
LuxD
LuxA
LuxB
Wavelength Scanning
A75G
LuxA
C106V
A75G
LuxA
A75G
V173A
LuxA
C106V
A75G V173A
LuxA
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
National Security
Code
486nm
488
489
490
492
600
Emission Peaks
500
400
300
200
0
100
200
300
400
500
600
700
Mutation
pSB417
A75G-V173A
PSB417
Intensity (a.u.)
A75G-C106V-V173A
V173A-C106V
A75G-C106V
A75G
C106V
V173A
Figure 1: Wavelength vs. Intensity
for clone PSB417 on which
subsequent mutations were
preformed. Maximum wavelength
was detected at 486nm.
Peak Wavelength
486
486
488
488
489
490
490
492
Figure 2: Wavelength
corresponding to maximum
luminosity. Clone V173A
showed the largest change
in wavelength ~6nm.
-100
In Vivo Imaging
V173A
LuxA
Symbol
Q
R
S
T
U
V
W
X
Y
Z
1
2
3
4
5
6
Intensity Integration
PSB417 Intensity (a.u.)
LuxE
C106V
Code
AAA
AAC
AAG
AAT
ACA
ACC
ACG
ACT
AGA
AGC
AGG
AGT
ATA
ATC
ATT
CAA
Symbol
A
B
C
D
E
0
XbaI Removal
We are very interested in development of a
bacterial “health thermometer” through the
coupling biosensors with our mutated luciferases.
The bacterial health thermometer would be
capable of transmitting light at a certain peak
wavelength to a cheap in house camera.
If certain disease associated small molecules, detectable in the
gut, were not at the proper concentrations, the camera would be
alerted and an alarm would sound. Different wavelengths of
light could be assigned to different chemicals, allowing for a
wide range of detection specificities
Light
100
Red Shifted Library Construction
Health
DNA
Abstract
Through site directed mutagenesis emitted light
was red-shifted to provide a library of optical
messages and to optimize tissue penetration. A
maximum shift of 6 nm in the intensity peak was
observed. DH5α E. Coli with different red-shifed
luciferases were fed to low germ mice and were
imaged with a highly sensitive CCD camera. It was
observed that the luminescent bacteria moved
from the stomach to the lower digestive tract
where they grew in population after several hours.
Applications
1200000
1000000
800000
600000
Series1
Although our original goal was for use of Human
Encryption by spies not wanting to deliver
information electronically (but rather via their gut).
However, we soon realized that our system would
be far more effective at tagging and labeling
people, possibly without them even knowing it.
Airports already use infrared to look for outbreaks (passengers
with fevers). It would be a very small step to put a filter over a
cheap CCD camera to measure peak wavelengths emitted from
the gut. Those with “dangerous” wavelengths could be brought
aside and searched. If you wanted to get cute you could also
include a message in the bacteria, using our DNA encryption
scheme, that might read, “this person was in Afghanistan on such
and such a date and is thought to be connected to this group.”
That message could be recovered via PCR of the stool followed
by sequencing with predesignated primers..
400000
200000
0
C106V
A75G
V173A
PSB417
Figure 3: After round 1 mutations
intensity is diminished from the
original clone PSB417 (relative
intensity on Y axis). Luminescence is
virtually eradicated in mutation
C106V but is restored as predicted by
protein structural analysis upon
subsequent mutations (data not
shown).
LuxA
Conclusion
Genetic engineering, biophotonics, and DNA sequencing have
enabled us to use mutalistic bacteria to transmit relevant
information. To our knowledge, these efforts represent the first
attempt to red-shift any bacteria luciferase capable of proper
functioning in mammals. It is interesting to note that we found
results are consistent with models of homologous luciferases which
are most active at 30C. Elaboration of our work could potentially
provide useful for applications beyond the vision of this project,
such as an improved structural/functional understanding of
luciferase and better tools to discover new antibiotics.
Acknowledgements:
C106V
V173A
Jingdong Tian (Duke University)
•Mike Winson (University of Wales Aberystwyth)
•Ashutosh Chilkoti (Duke University)
LuxA
Time=0
Time =17 hours
Figure 4: In vivo detection of luminescence conferred by PSB417.
Luminescence at time of administration (left) and next day (right).