Transcript Programmed population control by cell-cell

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
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
1
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.
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
2
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”
lacZα-ccdB, which is a fusion protein
(referred to as E in the next slide).

The lacZα part allows fusion protein
levels to be measured with a LacZ assay

The ccdB part kills susceptible cells by
poisoning the DNA gyrase complex
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
3
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.

Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
4
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
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
5
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.

Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
6
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

population control by cell-cell
Insets show the ON data on a linear scale. Programmed
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
7
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)

Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
8
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.
Programmed population control by cell-cell

communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
9
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.
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
10
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.
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
11
References

You, Lingchong et al. Programmed
population control by cell-cell
communication and regulated killing.
Letters to Nature 428 (2004)
Programmed population control by cell-cell
communication and regulated killing.You,
Lingchong et al. Letters to Nature 428
(2004)
12