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Ron Weiss |
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Tom Knight |
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MIT Artificial Intelligence Laboratory |
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phototropic or magnetotropic response |
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control of flagellar motors |
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chemical sensing and engineered enzymatic
release |
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selective protein expression |
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molecular scale fabrication |
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selective binding to membrane sites |
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collective behavior |
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autoinducers |
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slime molds |
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pattern formation |
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Cellular robotics requires |
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Intracellular control circuits |
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Intercellular signaling |
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First, characterize communication components |
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Engineer coordinated behavior using
diffusion-based communications |
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Previous Work |
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Implementing computation & communications |
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Intracellular regulation of transcription |
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Intercellular regulation of protein activity |
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Quorum sensing |
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Experimental Results |
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Conclusions |
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Cellular gate technology
[Knight & Sussman, ’98] |
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Simulation & characterization of gates and
circuits [Weiss, Homsy, Knight, ’98, ’99] |
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Toggle Switch implementation
[Gardner & Collins, ’00] |
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Ring Oscillator implementation
[Elowitz & Leibler, ’00] |
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With these inverters, any (finite) digital
circuit can be built |
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Inducers can inactivate repressors: |
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IPTG (Isopropylthio-ß-galactoside) à Lac
repressor |
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aTc (Anhydrotetracycline) à Tet
repressor |
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Use as a logical gate: |
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Inducers can also activate activators: |
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VAI (3-N-oxohexanoyl-L-Homoserine lacton) à luxR |
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Use as a logical (AND) gate: |
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Inducers and Co-repressors are termed effectors |
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Reasons to use effectors: |
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faster intracellular interactions |
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intercellular communications |
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Certain inducers useful for communications: |
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A cell produces inducer |
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Inducer diffuses outside the cell |
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Inducer enters another cell |
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Inducer interacts with repressor/activator à change
signal |
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Cell density dependent gene expression |
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Example:
Vibrio fischeri [density
dependent bioluminscence] |
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BIO-TEK FL600
Microplate Fluorescence Reader |
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Costar Transwell microplates
and cell culture inserts with
permeable membrane (0.1μm
pores) |
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Cells separated by function: |
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Sender cells in the bottom well |
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Receiver cells in the top well |
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Top excitation and emission fluorescence
readings |
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Figure shows fluorescence response of receiver
(pRCV-3) |
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Several cultures grown seperately overnight @37°C |
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Cultures mixed in 5 different ways and incubated
in FL600 @37°C |
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Fluorescence readings taken every 5 minutes for
2 hours |
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Figure shows response of receiver to different
levels of VAI |
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VAI extracted from pTK1 culture |
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Receiver cells (pRCV-3) grown @37°C to late log
phase |
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Receiver cells incubated in FL600 for 6 hours
@37°C with VAI |
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Data shows max fluorescence for each different
VAI level |
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Figure shows ability to induce stronger signals
with aTc |
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Non-induced sender (pLux8-Tet-8) & receiver
cells grown seperately @37°C to late log phase |
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Cells were combined in FL600, and sender cells
were induced with aTc |
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Data shows max fluorescence after 4 hours @37 °C
for 5 separate cultures plus control
[positive cultures have same DNA à variance due to OD] |
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This work: |
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Isolated an important intercellular
communications mechanism |
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Analyzed its components |
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Engineered its interfaces with standard genetic
control and reporter mechanisms |
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Future: |
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Additional analysis of lux characteristics |
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Examine and incorporate additional, non-cross
reacting, communications systems |
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Integrate communications with more sophisticated
in-vivo circuits |
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Engineer coordinated behavior (e.g. to form
patterns) |
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