IGEM:Cambridge/2008/Notebook/Turing Pattern Formation

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* contains erm insertion at amyE locus, so transformants at amyE locus can be screened for erythromycin resistance
* contains erm insertion at amyE locus, so transformants at amyE locus can be screened for erythromycin resistance
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=== Integration Vectors ===
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=== [[IGEM:Cambridge/2008/Turing Pattern Formation/Vectors]]| Vectors ===
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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.
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==== Ectopic Integration ====
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The integration site of these is pre-determined
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{| class="wikitable" border="1"
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|-
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! BGSC Accession
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! Vector Name
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! Features
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! Integration Locus
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! Vector Map
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! Vector Sequence
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|-
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| ECE112
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| pDG1661
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|
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* Chloramphenicol selectable in EC and BS
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* Spectinomycin selectable only in EC
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* spoVG-lacZ fusion: contains spoVG RBS
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* insert must contain a promoter
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* cassette insertion
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| amyE
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| map
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| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=1185558 U46196]
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|-
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| ECE171
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| pPyr-Kan
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|
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* Kanamycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* cassette insertion
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| pyrD
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| map
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| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726213 AY464559]
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|-
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| ECE172
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| pGlt-Cm
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|
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* Cloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* cassette insertion
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| gltA
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| map
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| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726216 AY464560]
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|-
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| ECE176
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| pBCJ164
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|
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* Cloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* cassette insertion
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* contains strong rspD promoter and RBS
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| rspD
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| map
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| [http://www.bgsc.org/sequences/pBCJ164.htm BGSC sequence]
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|-
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| ECE153
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| pSG1154
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|
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* Spectinomycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* cassette insertion
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* xylose-inducible promoter
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* high fluorescence GFP variant for protein fusions
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| amyE
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| map
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| not available
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|-
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| ECE162
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| pSG1193
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|
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* Spectinomycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* YFP full coding sequence, including RBS and Pxyl
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* xylose-inducible
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* cassette insertion
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| amyE
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| map
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| not available
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|-
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|}
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==== Insert Integration ====
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The user inserts a homologous piece of the Bacillus chromosome and the vector integrates there.
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{| class="wikitable" border="1"
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|-
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! BGSC Accession
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! Vector Name
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! Features
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! Vector Map
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! Vector Sequence
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|-
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| ECE149
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| pMUTIN-GFP+
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|
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* Erythromycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* GFP+ full coding sequence, including RBS and Pspac
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* IPTG-inducible
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* full plasmid insertion
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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutingfp.htm From Bayreuth]
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|-
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| ECE150
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| pMUTIN-CFP
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|
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* Erythromycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* CFP full coding sequence, including RBS and Pspac
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* IPTG-inducible
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* full plasmid insertion
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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincfp.htm From Bayreuth]
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|-
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| ECE151
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| pMUTIN-YFP
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|
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* Erythromycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* YFP full coding sequence, including RBS and Pspac
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* IPTG-inducible
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* full plasmid insertion
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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutinyfp.htm From Bayreuth]
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|-
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| ECE147
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| pMUTIN-cMyc
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|
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* Erythromycin selectable in EC or BS
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* Ampicillin selectable only in EC
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* cMyc antibody tag fusion
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* IPTG-inducible Pspac promoter
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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincmyc.htm From Bayreuth]
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|}
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=== Shuttle Vectors ===
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{| class="wikitable" border="1"
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|-
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! BGSC Accession
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! Vector Name
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! Features
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! Vector Map
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! Vector Sequence
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|-
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| ECE165
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| pAD123
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|
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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
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| map
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| [http://www.bgsc.org/sequences/pad123.htm BGSC sequence]
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|-
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| ECE166
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| pAD43-25
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|
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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
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| map
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| [http://www.bgsc.org/sequences/pad43-25.htm BGSC sequence]
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|-
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| ECE188
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| pHCMC02
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|
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* Chloramphenicol selectable in EC or BS
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* Ampicillin selectable only in EC
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* 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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|-
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| ECE189
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| pHCMC04
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|
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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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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04.htm]
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|-
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| ECE190
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| pHCMC05
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|
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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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| map
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| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC05.htm]
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|}
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2 shuttle vectors:
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*ppL82 (ampicillin) in DH5a
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*'''[[User:Daniel Goodman|Daniel Goodman]] 08:37, 25 July 2008 (UTC)''':check 2007 wiki to see what this is really
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*pNZ8901 (SURE plasmid, chloramphenicol) in MC1061
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*'''[[User:Daniel Goodman|Daniel Goodman]] 10:00, 22 July 2008 (UTC)''': See paper below on SURE expression system
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*'''[[User:Daniel Goodman|Daniel Goodman]] 10:00, 22 July 2008 (UTC)''': Can we get/do we have sequences of these?
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Revision as of 12:56, 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

IGEM:Cambridge/2008/Turing Pattern Formation/Vectors| Vectors

Research & Resources Page

Experiments

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