IGEM:Cambridge/2008/Turing Pattern Formation/Vectors

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All these vectors were kindly provided by Dr. Dan Ziegler, Director of the Bacillus Genetic Stock Center at Ohio State University.

Contents

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 Possible Uses Necessity (1-5 stars) Checked on Gel
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
ECE112 Map
ECE112 Map
U46196
  • Quantitative testing promoters using Beta-galactosidase assay
  • Biobricking: Has multiple pstI sites, Xba is not in cassette
**** Yes
ECE171 pPyr-Kan
  • Kanamycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
pyrD
ECE171 Map
ECE171 Map
AY464559
  • Inserting genes at neutral site
  • Can be used for biobrick insertion, all sites within cassette, PstI site is not at MCS
**** Yes
ECE172 pGlt-Cm
  • Cloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
gltA
ECE172 Map
ECE172 Map
AY464560
  • Inserting genes at neutral site
  • 4 pstI sites, EcoRI site, no Xba site
*: can use PyrD & AmyE instead No: wrong size
ECE176 pBCJ164
  • Cloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • cassette insertion
  • contains strong rspD promoter and RBS
rspD
ECE176 Map
ECE176 Map
BGSC sequence
  • Inserting genes at neutral site
  • Using strong rspD promoter
  • 2 pstI sites, X and S before promoter, E after terminator
*: use of strong rpsD promoter, but hard to insert into Yes
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
ECE153 Map
ECE153 Map
not available
  • GFP-fusion tracking of proteins (for agr components?)
  • multiple Xbas, multiple pstIs
***: inducible promoter, strong gfp, no sequence yes
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
ECE162 Map
ECE162 Map
not available
  • GFP-fusion tracking of proteins (for agr components?)
  • multiple Xbas, multiple pstIs
***: inducible promoter, strong gfp, no sequence inconclusive : only one band

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 Checked on gel
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
ECE149 Map
ECE149 Map
From Bayreuth No: 3 bands on the gel
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
ECE150 Map
ECE150 Map
From Bayreuth
(sequence incomplete)
yes
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
ECE151 Map
ECE151 Map
From Bayreuth Yes
ECE147 pMUTIN-cMyc
  • Erythromycin selectable in EC or BS
  • Ampicillin selectable only in EC
  • cMyc antibody tag fusion
  • IPTG-inducible Pspac promoter
ECE147 Map
ECE147 Map
From Bayreuth yes

Shuttle Vectors

BGSC Accession Vector Name Features Vector Map Vector Sequence Checked on gel
ECE165 pAD123
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • gfpmut3a in front of MCS, no promoter, but gram-positive RBS
ECE165 Map
ECE165 Map
BGSC sequence Yes
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
ECE166 Map
ECE166 Map
BGSC sequence Yes
ECE188 pHCMC02
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • weak constitutive PlepA promoter in front of MCS
ECE188 Map
ECE188 Map
From Bayreuth Not yet checked, DNA stocks
ECE189 pHCMC04
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • strong xylose inducible PxylA promoter in front of MCS
ECE189 Map
ECE189 Map
From Bayreuth Not yet checked, DNA stocks
ECE190 pHCMC05
  • Chloramphenicol selectable in EC or BS
  • Ampicillin selectable only in EC
  • strong xylose inducible PxylA promoter in front of MCS
ECE190 Map
ECE190 Map
From Bayreuth Yes


Plasmid Gels

  • We ran these plasmids on gels, using double and single digests, to check them
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