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Bacterial Edge Detector UT AUSTIN / UCSF IGEM 2006. Victoria hsiao . Charles Darwin, immortalized in E.coli . 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.

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bacterial edge detector ut austin ucsf igem 2006

Bacterial Edge DetectorUT AUSTIN / UCSF IGEM 2006

Victoria hsiao

Charles Darwin, immortalized in E.coli

http://parts.mit.edu/wiki/index.php/University_of_Texas_2006

projects mentioned in the presentation
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.

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

engineering a bacterial edge detector

Engineering a Bacterial Edge Detector

iGEM 2006

UT Austin/UCSF

Lawn of E.coli

Printed transparency

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

steps to engineering a bacterial edge detector
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)

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

background bacterial photography 2005
Background: Bacterial Photography (2005)
  • 1. Getting E.coli to detect light
  • Make Cph1 (photoreceptor) from heme by using the genes ho1 and pcyA (derived from cyanobacteria)
  • 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
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.

Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006

light imaging setup
Light Imaging Setup

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
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

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

edge detection 2006
Edge Detection (2006)
  • 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
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 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
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 .

Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006

edge detection logic continued1
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..

Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006

leaky c1 light repression
Leaky C1 light repression

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
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.

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

it worked
It Worked!

Images: http://parts.mit.edu/wiki/index.php/University_of_Texas_2006

improving contrast sharpness of edge detection
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 > 7.5)

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

other things they found
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.

“Spatial Recognition of Bacterial Populations” UT Austin/ UCSF iGEM 2006 Presentation Ppt

things i thought were exciting
Things I Thought Were Exciting
  • E.coli can be madeinto 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
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/wiki/index.php/University_of_Texas_2006