Programmed population control by cell-cell communication and regulated killing

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Programmed population control by cell-cell
communication and regulated killing

• Lingchong You, Robert Sidney Cox III, Ron Weiss
  Frances H. Arnold

Presented by Victoria Hsiao
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What They Did

• They built and characterized a population
  control circuit which can automatically
  regulate the density of an E.coli population.
• Quorum sensing- when bacteria regulate gene
  expression based on population density (which
  they sense based on the density of signaling
  molecules).
• Negative feedback loop Bacteria produce
  signaling molecule ? as of bac increases, so
  does the density of the signal ? at a certain
  threshold, the quorum sensing kicks in, which
  leads to cell death.

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The Plasmids

• pLuxR12 contains the genes for the LuxR/LuxI
  system from the marine bacterium V. fischeri.
• LuxI ?The LuxI protein, which makes
  acyl-homoserine lactone (AHL) a small
  diffusible signaling molecule.
• LuxR ? LuxR transcriptional regulator ? when
  activated with AHL, induces the expression of the
  killer gene
• pluxCcdB3 contains the killer gene lacZa-ccdB,
  which is a fusion protein (referred to as E in
  the next slide).
• The lacZa part allows fusion protein levels to be
  measured with a LacZ assay
• The ccdB part kills susceptible cells by
  poisoning the DNA gyrase complex

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The Circuit

• LuxI protein produces AHL, which accumulates in
  the medium and inside the cells.
• Once the AHL reaches a high enough concentration,
  it will bind and activate the LuxR
  transcriptional regulator, which binds to the
  luxI promoter.
• E, the killer protein, is then expressed and
  causes cells death at high enough levels.

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Mathematical Model

• N (mL-1) viable cell density
• k (h-1) growth rate
• Nm (mL-1) carrying capacity
• E (nM) concentration of killer protein
• d (nM-1h-1) death rate constant
• A concentration of AHL
• kE dE growth and degradation rate constants
  of E
• vA dA same for AHL

Eq. 1) Cell Growth and Death
Eq. 2) Production and Degradation of the Killer
Protein
Eq. 3) Production and Degradation of AHL
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Mathematical Model

• Using the mathematical model, Arnold et al.
  predicted that the system would reach a stable
  cell density for all realistic parameter values,
  though it may or may not have damped oscillations
  while going to steady state.
• Predicts that steady-state density increases
  proportionally with the AHL degradation rate
  constant.

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Experimental Results

• Culture with circuit OFF
• Culture with circuit ON
• CFU colony-forming units (per mL)
• LacZ activity shows how much killer protein is
  being expressed
• Insets show the ON data on a linear scale.

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Controlling steady-state density with AHL

• To confirm that the killer protein production
  rate was limited by AHL production in the ON
  circuit, 200mL of exogenous AHL were added to the
  media.
• As expected, it did not affect bacteria without
  the circuit or with the OFF, but prevented growth
  completely in the bacteria with the ON circuit.
• They were able to change the steady-state
  density of the E.coli population by using AHL
  degradation rate as a dial.
• The AHL degradation rate was controlled by
  changing the pH of the medium (?pH ?Ns)

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Effects of pH on circuit behavior

• Steady state density increases as pH increases
• Levels of killer protein (as shown by the lacZ
  activity) remained roughly the same despite
  changes in pH. (this is predicted in their model
  if you take Eq. 1 and solve for E with dN/dt 0
  ? Es k/d)
• (e) shows that normalized Ns has a nearly linear
  dependence on pH.
• (j) shows that killer protein expression remains
  nearly constant despite changes in pH.

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The big ideas

• Using cell-cell communication to coordinate
  behavior across the population.
• Population-control circuits that have cell-cell
  communication actually require phenotypic
  variation to work.
• This way, cells have different tolerances for the
  killer protein. Otherwise, if all the bacteria
  had the same phenotype, then once the killer
  protein reached a critical density, all the cells
  would die.

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Discussion

• Liked
• Self-regulating system based on a single negative
  feedback loop. I liked the idea that once you
  added the plasmids, you could sort of stand back
  and see what happens.
• Worked the best with phenotypic variation. I
  just thought it was cool that the system
  accounted for, and actually depended on, genetic
  variation in the bacteria.
• The final steady-state density can be tuned by
  changing the pH of the medium, which seems like a
  simple and easy way to set the final state of the
  system.
• Disliked
• Bacteria already have this sort of feedback loop
  in response to crowding/nutrient depletion, so
  while it was cool that they could change the
  environmental cue that triggered cell death, it
  also seemed sort of basic.
• But this is a foundational paper, which is
  supposed to lead to building synthetic ecosystems
  with programmed interactions between bacterial
  communities.

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References

• You, Lingchong et al. Programmed population
  control by cell-cell communication and regulated
  killing. Letters to Nature 428 (2004)