Transcript Bacterial Edge Detector

Bacterial Edge Detector
UT AUSTIN / UCSF
IGEM 2006
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VICTORIA HSIAO
Charles Darwin,
immortalized in
E.coli 
http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Projects mentioned in the presentation
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 Bacterial Edge Detector – engineering E.coli to detect
light/dark boundaries and make a black outline only on
those edges.
 Regulating chemotaxis in E.coli – engineering E.coli
to “tumble” in place instead of swimming straight, thus
making them stationary.
 Photofabrication – using mask-directed lithography
to make 3D microstructures from cross-linked proteins
that living organisms can interact with.
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006
Presentation Ppt
Engineering a Bacterial Edge
Detector
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IGEM 2006
UT AUSTIN/UCSF
Printed
transparency
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
Lawn of E.coli
Steps to Engineering a Bacterial Edge Detector
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 1. Make E.coli (which is blind) able to detect light
(iGEM 2005)
 2. Capture an image with a lawn of E.coli
(iGEM 2005)
 3. Make the E.coli compute the light/dark boundary
of the captured image
(iGEM 2006)
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
Background: Bacterial Photography (2005)
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 1. Getting E.coli to detect light
1. Make Cph1
(photoreceptor)
from heme by
using the genes
ho1 and pcyA
(derived from
cyanobacteria)
2. Cph1 is combined
with the histidine
kinase Env Z
(which e.coli
already had) to
make the chimera
Cph8.
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Photography: Light vs Dark
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In the dark, the Env Z phosphorylates
the OmpR transcription factor protein,
which binds to the promoter PompC,
which expresses the reporter LacZ,
which will produce a black precipitate
when the sugar S-gal is added.
When the E.coli is exposed to 660 nm
light, the transcription cascade is
repressed because Cph 1 isomerizes and
changes its conformation, which
inactivates the Env Z. Thus, these
sections remain light.
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Light Imaging Setup
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Mercury lamp
632nm bandpass
filter
35mm slide
Double
Gauss
focusable
lens
Projected Image
“Engineering Escherichia Coli to see light” Levskaya et al. Nature, Brief Communications 2005 (Supplementary Materials)
Steps 1 & 2 Completed
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• Light areas are light, dark areas are dark, but can we make an outline?
(2005) The E.coli could make a highcontrast replica of the projected image
(2006) Step 3 : taking the projected image and only
showing expressing black at the edges of light and
dark
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
Edge Detection (2006)
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 Transcription cascade is “black-boxed” into an
inverter block
Red light
Gene
expression
repressed!
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
The Edge Detection Circuitry
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Lux 1 gene – lacZ
activator, produces
AHL (acylated
homoserine lactone)
which binds to LuxR.
C1 gene – lacZ
dominant repressor,
produces c1 which
binds to Oλ , repressing
lacZ even if lux1 is
activated.
Therefore, the only way
to get a black output is
to have AHL, but while
c1 is repressed. How?
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Edge Detection Logic (continued)
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Dark
Light
In both cases, light and dark, lacZ expression is repressed.
However, the AHL produced by the dark bacteria is able to diffuse to
surrounding bacteria, and only the light bacteria will be able to use it .
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Edge Detection Logic (continued)
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Therefore, this case can
only occur in light
bacteria at the light/dark
boundary, and the E.coli
can detect edges..
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Leaky C1 light repression
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When light was projected onto the E.coli
the C1 gene wasn’t being entirely
repressed, so when the AHL diffused over
from the dark bacteria, lacZ was still
repressed.
Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Toning Down C1 Expression with RBS
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By adding ribosomal
binding sites (RBS) to the
gene sequence, they were
able to tone down the
expression of C1 such that
it was still dominant in
the dark, but permissive
in the light.
So they tried 3 different
concentrations of RBS:
RBS3 0.07x was the only
one that worked.
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
It Worked!
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Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006
Improving Contrast & Sharpness of Edge
Detection
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 LuxI poison that is expressed in light so that there is
less of a gradient at the edge.
 Mix in Aiia (anti-AHL) expressing strain to take up
AHL at different rates to alter the width of the edge.
 Modify pH (AHL is destabilized by > 7.5)
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
Other things they found
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 In all the experiments just described they used a two plasmid
system to transform the E.coli:


Plasmid 1 contained the phycobilins ho1 and pcyA (which make
photoreceptor in Cph1)
Plasmid 2 contained Cph8
 When they combined the two plasmids into a single plasmid
(Bba_M30109), they got inverted logic. So now, light
activated both luxI and ch1 while dark repressed. But then
they noted that the background signal of Bba_M30109 was
too high for bacterial photographs.
 They also found that Cph1 responds to another wavelength in
addition to the 660nm. A 735nm wavelength changes the
Cph1 conformation in such a way that the dark conditions are
activated. This is useful because a 735nm light can sometimes
be aimed more precisely than a projected shadow.
“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt
Things I Thought Were Exciting
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 E.coli can be made into light sensors just by
combining photoreceptor genes from cyanobacteria
with genes for an enzyme that E.coli already has.
 Making each cell do a simple computation, so that
having an entire lawn of bacteria results in massive
parallel computations.
Sources
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 “Engineering Escherichia Coli to see light” Levskaya
et al. Nature, Brief Communications 2005
(Supplementary Materials)
 “Spatial Recognition of Bacterial Populations” UT
Austin/ UCSF iGEM 2006 Presentation Ppt
 UT Austin iGEM Wiki page,
http://parts.mit.edu/wiki/index.php/University_of_
Texas_2006