# IGEM:Cambridge/2008/Notebook/Turing Pattern Formation

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== Materials == | == Materials == | ||

- | Bacillus strain 1A1 (derivative of strain 168) | + | === Bacillus Strains === |

+ | |||

+ | Bacillus strain 1A1 (derivative of standard strain 168) | ||

* deficient in tryptophan, have to add to media | * deficient in tryptophan, have to add to media | ||

* keep at room temp, aren't freezable | * keep at room temp, aren't freezable | ||

+ | |||

+ | Bacillus strain 1A771 (derivative of standard strain 168) | ||

+ | * deficient at tryptophan, have to add to media | ||

+ | * keep at room temp, aren't freezable | ||

+ | * contains erm insertion at amyE locus, so transformants at amyE locus can be screened for erythromycin resistance | ||

+ | |||

+ | === Integration Vectors === | ||

+ | These vectors integrate into the chromosome and do not have a replication origin in Bacillus. They either integrate cassettes that require double crossovers and two homologous regions, or whole-plasmid insertions that only require one crossover and one corresponding region of homology. | ||

+ | |||

+ | ==== Ectopic Integration ==== | ||

+ | The integration site of these is pre-determined. | ||

+ | |||

+ | ==== Insert Integration ==== | ||

+ | The user inserts a homologous piece of the Bacillus chromosome and the vector integrates there. | ||

+ | |||

+ | |||

+ | |||

+ | === Shuttle Vectors === | ||

+ | |||

+ | |||

## Revision as of 10:50, 31 July 2008

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## IntroductionWe are planning to implement a simple two-component Reaction-Diffusion system in the gram-positive model organism Bacillus subtilis. In 1952, Alan Turing famously described this system and suggested it as the basis for self-organization and pattern formation in biological systems. The simplest of these patterns, which we are planning to model in bacteria, mimic the spots and stripes seen on animal coats.
(A) The model consists of two diffusible signals secreted by every cell. The activator, which is controlled by a stochastic bistable switch, turns on itself and its own inhibitor. (B) A field of cells can be stably patterned into two different zones, so long as the inhibitor diffuses faster than the activator. The activator and inhibitor are synthesized in the source at the center, and turned off by accumulation of the inhibitor in the periphery.
We plan to use two well-characterized bacterial communication systems to generate this behavior. The agr peptide signalling system from S. aureus will serve as our activatory signal (pictured), while the lux system from V. fischeri will serve as our inhibitor. Bacillus subtilis serves as an excellent chassis for this project because of the ease with which chromosomal integration can be performed. This project will focus on a tight integration of modeling and experiment; we will test different promoter strengths and other variables, feed these system parameters into our multi-cell models, and then use those models to tweak the regulatory machinery that will control signal production. ## Grasshopper ExampleThe reaction-diffusion system depends on an activator and inhibitory signal that spread throughout the medium. The "grasshopper" example is quite intuitive: Imagine it is hot and there is a field of dry grass with grasshoppers. Suddenly, a fire starts burning at some point and spreads (the activator signal) so that the grasshoppers move away from that point to avoid the fire. However, the grasshoppers also generate moisture (the inhibitory signal) thus preventing the areas of dry grass the grasshoppers move to of catching fire. The result will be the initial patch of the field that has burnt down surrounded by moisture preventing the fire from spreading. Imagine now that at the beginning, not a single place but numerous randomly distributed places (resembling noise) of dry grass caught fire. The resulting patterning of charred grass and grasshoppers is called a Turing Pattern. It is important to note is that the inhibitory signal (grasshoppers) must travel faster than the activation signal (fire) as to prevent the whole field from burning down.
## ObjectiveThis project seeks to generate Turing Patterns by creating a Reaction-Diffusion system in the gram-positive bacteria Bacillus subtilis. We need to integrate two signalling systems into this bacterium and use an autofeedback mechanism to generate self-organizing patterns from random noise. We plan to incorporate the
## Materials## Bacillus StrainsBacillus strain 1A1 (derivative of standard strain 168) - deficient in tryptophan, have to add to media
- keep at room temp, aren't freezable
Bacillus strain 1A771 (derivative of standard strain 168) - deficient at tryptophan, have to add to media
- keep at room temp, aren't freezable
- contains erm insertion at amyE locus, so transformants at amyE locus can be screened for erythromycin resistance
## Integration VectorsThese vectors integrate into the chromosome and do not have a replication origin in Bacillus. They either integrate cassettes that require double crossovers and two homologous regions, or whole-plasmid insertions that only require one crossover and one corresponding region of homology. ## Ectopic IntegrationThe integration site of these is pre-determined. ## Insert IntegrationThe user inserts a homologous piece of the Bacillus chromosome and the vector integrates there.
## Shuttle Vectors2 shuttle vectors: - ppL82 (ampicillin) in DH5a
**Daniel Goodman 08:37, 25 July 2008 (UTC)**:check 2007 wiki to see what this is really
- pNZ8901 (SURE plasmid, chloramphenicol) in MC1061
**Daniel Goodman 10:00, 22 July 2008 (UTC)**: See paper below on SURE expression system**Daniel Goodman 10:00, 22 July 2008 (UTC)**: Can we get/do we have sequences of these?
## Research & Resources Page## Experiments |