IGEM:Cambridge/2008/Notebook/Turing Pattern Formation

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| ECE165
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| pAD123
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* Chloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* gfpmut3a in front of MCS, no promoter, but gram-positive RBS
| map
| map
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| seq link
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| [http://www.bgsc.org/sequences/pad123.htm BGSC sequence]
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| ECE166
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| pAD43-25
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* Chloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* gfpmut3a in front of MCS, strong Pupp promoter, gram-positive RBS
| map
| map
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| seq link
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| [http://www.bgsc.org/sequences/pad43-25.htm BGSC sequence]
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| ECE188
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| vector name
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| pHCMC02
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* Chloramphenicol selectable in EC or BS
 +
* Ampicillin selectable only in EC
 +
* weak constitutive PlepA promoter in front of MCS
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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC02.htm]
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| ECE189
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| pHCMC04
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* Chloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* strong xylose inducible PxylA promoter in front of MCS
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04.htm]
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| ECE190
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* Chloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* strong xylose inducible PxylA promoter in front of MCS
| map
| map
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| seq link
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC05.htm]
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Revision as of 12:54, 31 July 2008



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Introduction

We 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.

2kgl.gif

struktur-e.gif b701571b-f1.gif

(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.

Cambridge_Agr_operon_and_biochemical_pathways.png

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 Example

The 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.


Objective

Schematic of Activator/Inhibitor system in B. subtilis
Schematic of Activator/Inhibitor system in B. subtilis

This 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 agr peptide signalling system from S. aureus and the lux AHL system from V. fisheri.


Materials

Bacillus Strains

Bacillus 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 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

BGSC Accession Vector Name Features Integration Locus Vector Map Vector Sequence
ECE112 pDG1661
  • Chloramphenicol selectable in EC and BS
  • Spectinomycin selectable only in EC
  • spoVG-lacZ fusion: contains spoVG RBS
  • insert must contain a promoter
  • cassette insertion
amyE map U46196
ECE171 pPyr-Kan
  • Kanamycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
pyrD map AY464559
ECE172 pGlt-Cm
  • Cloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
gltA map AY464560
ECE176 pBCJ164
  • Cloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
  • contains strong rspD promoter and RBS
rspD map BGSC sequence
ECE153 pSG1154
  • Spectinomycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
  • xylose-inducible promoter
  • high fluorescence GFP variant for protein fusions
amyE map not available
ECE162 pSG1193
  • Spectinomycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • YFP full coding sequence, including RBS and Pxyl
  • xylose-inducible
  • cassette insertion
amyE map not available

Insert Integration

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

BGSC Accession Vector Name Features Vector Map Vector Sequence
ECE149 pMUTIN-GFP+
  • Erythromycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • GFP+ full coding sequence, including RBS and Pspac
  • IPTG-inducible
  • full plasmid insertion
map From Bayreuth
ECE150 pMUTIN-CFP
  • Erythromycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • CFP full coding sequence, including RBS and Pspac
  • IPTG-inducible
  • full plasmid insertion
map From Bayreuth
ECE151 pMUTIN-YFP
  • Erythromycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • YFP full coding sequence, including RBS and Pspac
  • IPTG-inducible
  • full plasmid insertion
map From Bayreuth
ECE147 pMUTIN-cMyc
  • Erythromycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • cMyc antibody tag fusion
  • IPTG-inducible Pspac promoter
map From Bayreuth

Shuttle Vectors

BGSC Accession Vector Name Features Vector Map Vector Sequence
ECE165 pAD123
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • gfpmut3a in front of MCS, no promoter, but gram-positive RBS
map BGSC sequence
ECE166 pAD43-25
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • gfpmut3a in front of MCS, strong Pupp promoter, gram-positive RBS
map BGSC sequence
ECE188 pHCMC02
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • weak constitutive PlepA promoter in front of MCS
map [1]
ECE189 pHCMC04
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • strong xylose inducible PxylA promoter in front of MCS
map [2]
ECE190 pHCMC05
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • strong xylose inducible PxylA promoter in front of MCS
map [3]


2 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?


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