IAP2004:Polkadorks

From 2006.igem.org

Polkadorks and the E. Coli-brator

est. January 5, 2004

Dorks: Ziyan Chu, Roshan Kumar, Stephen Lee, Joe Levine

Background

Last year's groups were asked to create systems that blink; this year our job was to create systems that form a spatial design. We, the Polkadorks, chose polkadots for our design and have spent the month of January 2004 working on this system.

System Overview

This animation illustrates our basic system. We start with a collection of engineered e-coli moving randomly in plated media. The bacteria are represented by black dots in the animation. Under control of a stochastic element, a few begin excreting an attractant. These bacteria we will call the sender cells. In the movie we have one sender cell represented by the red dot; the green circle represents the attractant diffusing away from the sender cell.

Through chemotaxis, a process by which a cell along a chemical gradient swims toward or away from the stimulus (an attractant in this case), nearby bacteria start swimming towards the sender cells. These bacteria we will call the receiver cells. This way, groups begin forming around the original sender cells on the plate.

All bacteria have been engineered with a quorum sensing mechanism which effectively senses local cell density. In the groups that have formed on our plate, the cell density eventually reaches a certain threshold. The quorum sensing mechanism of the cells then stops secretion of any attractant. The existing attractant then diffuses away. Since there is no more attractant being secreted, the cells will diffuse away and eventually be spread out across the plate once again. Then by the stochastic element a few cells will begin excreting the attractant and the whole process is repeated.

Basically polkadots will form, diffuse, and form again in random areas on the plate. Our system should thus form time-varying patterns based on local random time-varying symmetry breaking.

Device-Level System Diagram

Parts-Level Device & System Diagram

Layout Diagram

Timing Diagram

Shown is a putative timing diagram of our system for a typical attractant cycle. At some time the trigger activates randomly, sending a TIPS signal to the switch. The switch output rises with a delay set by protein synthesis and degredation (to be explained in the switch device section). This causes the AspA level to rise with another protein synthesis delay. The corresponding increase in Aspartate will activate the switches of other cells (receiver cell in the diagram). After an undefined attraction period, the cells will have formed a dense enough cluster to activate quorum sensing. The switch will turn off with a similar protein synthesis/degredation delay, and aspartate will begin to diffuse away. The cell cluster will diffuse away, at which point the system will be reset to its initial conditions.

Population Simulations

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Characterization

We have specified various protocols to validate our system part by part. We will validate each individual device in our block diagram separately and then subsequently test compound parts. Our plans for device testing are listed below.

Testing Single Devices

   *Trigger
   *Switch
   *Aspartate Sender
   *Aspartate Receiver
   *Quorum Sender
   *Quorum Receiver

Testing Compound Devices

   *Aspartate Sender & Switch
   *Aspartate Receiver & Switch
   *Quorum Receiver & Switch

Testing Collective Behaviors

   *Attractant-Chemotaxis Interaction
   *Characterizing Quorum Response to Clustering

Parts List

Systems Device	Device Name	Part Description	BioBricks Number
Trigger.1	Lac stochastic trigger device		BBa_I3100
		Constitutive Promoter	BBa_R1075
		RBS	BBa_B0034
		LacI CDS	BBa_C0012
		TT	BBa_B0015
		LacI Promoter	BBa_R0010
Receive.1	Tar-EnvZ attractant receiver device		BBa_I3300
		Constitutive Promoter	BBa_R1075
		RBS	BBa_B0034
		Tar-EnvZ CDS	BBa_C0082
		TT	BBa_B0015
		OmpR Promoter	BBa_R0082
Quorum Receive.1
Quorum receiver device			BBa_I3510
		Lux pL Promoter	BBa_R0063
		LuxR Device	BBa_I0462
		Lux pR Promoter	BBa_R0062
Switch.1
Switch In (On)	Hk022cI switch input device		BBa_I3400
		RBS	BBa_B0034
		Hk022cI CDS	BBa_C0050
		TT	BBa_B0015
Switch In (Off)	Lambda cI switch input device		BBa_I3401
		RBS	BBa_B0034
		Lambda cI CDS	BBa_C0051
		TT	BBa_B0015
Switch Out (On)	Hk022cI switch output device		BBa_I3410
		Promoter	BBa_R0051
		RBS	BBa_B0034
		hk022cI CDS	BBa_C0050
Switch Out (Off)	Lambda cI switch output device		BBa_I3411
		Promoter	BBa_R0050
		RBS	BBa_B0034
		Lambda cI CDS	BBa_C0051
Attract.1	AspA attractant sender device		BBa_I3200
		RBS	BBa_B0034
		AspA CDS	BBa_C0083
Report.1	CFP Reporter Device		BBa_E0422
		RBS	BBa_B0034
		CFP CDS	BBa_E0022
		TT	BBa_B0015
Quorum Send.1	Quorum sender device		BBa_I3500
		Lux pR Promoter	BBa_R0062
		LuxI Device	BBa_I0461

References

• Betterton MD and Brenner MP. Collapsing Bacterial Cylinders. Physical Review E v64 pp061904-061918.

• Brenner MP, Levitov LS, Budrene EO. Physical Mechanisms for Chemotactic Pattern Formation by Bacteria. Biophysical Journal. v74 1677-1693, April 1998.

• NBudrene EO, Berg HC. Dynamics of formation of symmetrical patterns by chemotactic bacteria. Nature 376, 49-53 (1995).

• Elowitz M and Liebler S. A synthetic oscillatory network of transcriptional regulators. Nature 403:335-338 (2000).

• Forst SA, Delgado J, Inouye M. DNA-binding properties of the transcription activator (OmpR) for the upstream sequences of ompF in Escherichia coli are altered by envZ mutations and medium osmolarity. Journal of Bacteriology. 171 (6), 2949-2955 (1989)

• Gardner TS, Cantor CR, Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature 403:339:342 (2000).

• Maeda S and Mizuno T. Evidence for multiple OmpR binding sites in the upstream activation sequence of the OmpC promoter in Excherichia coli: a single OmpR-binding site is capable of activating the promoter. Journal of Bacteriology. 172 (1), 501-503 (1990).

• Mittal N, Budrene EO, Brenner MP, van Oudenaarden A. Motility of Escherichia coli cells in clusters formed by chemotactic aggregation. Proceedings of the National Academy of Sciences, v100 n23, Nov 11 2003 pp 13259-13263.

• Utsumi R, Brissette RE, Rampersaud A, Forst SA, Oosawa K, Inouye M. Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate. Science v245 pp1246-1249 (1989)

• Weiss, R. Cellular Computation and Communications using Engineered Genetic Regulatory Networks. MIT EECS PhD Thesis. August, 2001.