Iap 2004

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Contents

Overview

During MIT's Independent Activity Period in January 2004, teams designed genetic systems to create cellular patterns varying from bull’s-eyes to polka dots and even dynamic designs where cells swim together. From these designs, standard biological parts were designed and synthesized.



Polkadorks and the E. Coli-brator

Frinksmiling.jpg

est. January 5, 2004




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.


Drew Endy, our fearless leader




Basic Design

Intro1-EcolibratorMovie.gif

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.



System Diagram

Intro21-SystemDiagram.jpg


Device Diagram

Intro22-DeviceDiagram.jpg


Layout Diagram

Intro23-LayoutDiagram.jpg


Timing Diagram

Intro3-TimingDiagram.jpg


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

Frinkburger.gif

_ _ 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

IAP 2004 Projects

Descriptions and schematics can be found [http://iap03.igem.org here]

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
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