Programmed population control by cell-cell communication and regulated killing

1. 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)

2. 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)

3. 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 lacZαpart 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)

4. 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)

5. 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) =deathrate 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)

6. 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)

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

8. 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)

9. 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)

10. 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)

11. 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)

12. 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)