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A synthetic Escherichia colipredator-prey ecosystem

Balagaddé et al. (2008)

Kathrin NußbaumiGEM Journal Club

A synthetic Escherichia coli predator-prey ecosystem 2

Idea

• Construct a synthetic ecosystem– 2 Escherichia coli populations– Communicate bi-directional through quorum sensing– Regulate each others gene expression and survival

• The predator kills the prey by – inducing expression of a killer protein

• The prey rescues the predator by– eliciting expression of an antidote protein in the predator

• Microchemostats used as long-term culturing of the system

• Simple mathematical model


Microchemostats

• Balagadde et al, 2005

• Chip-based bioreactor• Use microfluidic plumbing

networks to actively prevent biofilm formation

• Bioreactor enables long-term culture and monitoring of extremely small populations of bacteria – ~100 to ~104 bacteria

• Cultures monitored in situ by optical microscopy

2 mm

18 mm


Microchemostats

• Six independent 16-nanoliter reactors

• Each reactor consists of – a growth chamber

(fluidic loop) – an integrated

peristaltic pump– micromechanical valves to

- add medium - remove waste - recover cells

– 16 individually addressable segments

• Two altering states:– continuous circulation– cleaning and dilution

Cleaning and dilution


Microchemostats

Continuous circulation


Modified microchemostats

• Allow accurate monitoring of community composition


Basics

• Construction of synthetic gene circuit that converted two E..coli populations into a predator-prey system

• Differences to the canonical predator-prey system– Instead of acting as food source, the prey provides an

‘antidote’ to programmed death of the predator– There is competition between predator (MG1655 strain) and

prey (Top10F’ strain) for nutrients in a co-culture which is generally absent in natural predator-prey systems

• Quorum sensing modules were used to enable two-way communication between predator and prey populations– LuxI/LuxR from Vibrio fischeri– LasI/LasR from Pseudomonas aeruginosa


LuxR/LuxI and LasR/LasI

• Receiver 1– Encodes LuxR– Responded to

cells sending 3OC6HSL

• Receiver 2– Encodes LasI– Responds to

cells sending 3OC12HSL

Active LasR and active LuxR can induce gene expression at the luxI promotor (PluxI)


Synthetic predator-prey system


Prediction of the extinction, co-existence or oscillatory dynamics

Predator-prey oscillation

Prey domination

Predator domination


Growth behavior of predator

• Verification of predator function

OFF cultures – no inducers→ predator grew to a high

density

+IPTG cultures (ON)– 1 mM IPTG → predator growth was

significantly inhibited by IPTG, which induces the CcdBexpression

+IPTG +3OC6HSL cultures– 1 mM IPTG– 100 mM AHL→ predator growth can be

saved by supplementary AHL, which can initiate the CcdAexpression


Growth behavior of prey

• Verification of prey function

OFF cultures – no inducers→ prey grew to a high density

+IPTG cultures (ON)– 1 mM IPTG → prey growth was hardly

inhibited by IPTG

+IPTG +3OC12HSL cultures– 1 mM IPTG– 100 mM AHL→ prey growth was significantly

inhibited by supplementary AHL, which initiate the CcdBexpression


Oscillatory dynamics

• Typical oscillatory dynamics of predator and prey population measured in microchemostat

• When prey grew high→ predator growth

• When prey density was low→ predator growth

inhibited by constitutive CcdBexpression

• Oscillation resembles the natural predator-prey system


System dynamics –varying IPTG induction levels


Dependence of system dynamics on dilution rate (D)

• An increase in D leads to– a phase of damped

oscillatory dynamics– decrease in period of

oscillation


Summary

• Microchemostat is a high-throughput screening technology that can perform rapid characterization of synthetic circuits with long-term, non-invasive measurements of microbial population properties under steady-state conditions

• A synthetic ecosystem was constructed consisting of two E. colipopulations, which communicate bi-directional through quorum sensing

– Differences compared to a natural predator-prey system– The predator kills the prey by inducing expression of a killer protein– The prey rescues the predator by eliciting expression of an antidote protein in

the predator

• The system yielded oscillatory dynamics that resemble the natural predator-prey system

• Higher IPTG levels (≥ 5 μM) initiate oscillatory dynamics between the predator and the prey populations

• An increase in the dilution rate (D) can lead to a significant decrease in the period of oscillations and lead to a fast, damped oscillation


Summary

• This study represents a major advance beyond recent efforts in constructing synthetic ecosystems, in terms of system complexity and resolution of characterization

• It provides a framework for exploring how naturally existing cellular components can be assembled to program and coordinate highly complex cellular behavior

• The system allows direct manipulation of intrinsic parameters, such as growth rate, death rate and strength of cell–cell communication

• Synthetic ecosystems may serve as well-defined systems for exploring evolutionary and ecological questions regarding, for example, the generation and maintenance of biodiversity


References

1. Balagaddé FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. A synthetic Escherichia coli predator-preyecosystem. Mol Syst Biol. Epub 2008 Apr 15; 4:187.

2. Balagaddé FK, You L, Hansen CL, Arnold FH, Quake SR. Long-term monitoring of bacteria undergoing programmed populationcontrol in a microchemostat. Science. 2005 Jul 1;309(5731):137-40.

a synthetic final - main page - 2008.igem.org2008.igem.org/wiki/images/d/d8/pres_kat.pdf · ahl,...

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