Victoria hsiao

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Bacterial Edge DetectorUT AUSTIN / UCSF IGEM
2006

• Victoria hsiao

Charles Darwin, immortalized in E.coli ?
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Projects mentioned in the presentation

• 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.

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Engineering a Bacterial Edge Detector

• iGEM 2006
• UT Austin/UCSF

Lawn of E.coli
Printed transparency
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Steps to Engineering a Bacterial Edge Detector

• 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)

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Background Bacterial Photography (2005)

• 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.

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Photography Light vs Dark
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.
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Light Imaging Setup
Mercury lamp
632nm bandpass filter
35mm slide
Double Gauss focusable lens
Projected Image
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Steps 1 2 Completed

• Light areas are light, dark areas are dark, but
  can we make an outline?

(2005) The E.coli could make a high-contrast
replica of the projected image
(2006) Step 3 taking the projected image and
only showing expressing black at the edges of
light and dark
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Edge Detection (2006)

• Transcription cascade is black-boxed into an
  inverter block

Red light
Gene expression repressed!
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The Edge Detection Circuitry
Lux 1 gene lacZ activator, produces AHL
(acylated homoserine lactone) which binds to
LuxR. C1 gene lacZ dominant repressor,
produces c1 which binds to O? , 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?
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Edge Detection Logic (continued)
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 .
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Edge Detection Logic (continued)
Therefore, this case can only occur in light
bacteria at the light/dark boundary, and the
E.coli can detect edges..
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Leaky C1 light repression
When light was projected onto the E.coli the C1
gene wasnt being entirely repressed, so when the
AHL diffused over from the dark bacteria, lacZ
was still repressed.
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Toning Down C1 Expression with RBS
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.
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It Worked!
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Improving Contrast Sharpness of Edge Detection

• 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 gt 7.5)

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Other things they found

• 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.

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Things I Thought Were Exciting

• 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.

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Sources

• 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/wik
  i/index.php/University_of_Texas_2006