IGEM:Cambridge/2008/Turing Pattern Formation/Vectors: Difference between revisions

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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.
All these vectors were kindly provided by Dr. Dan Ziegler, Director of the Bacillus Genetic Stock Center at Ohio State University.
 
'''[[User:Daniel Goodman|Daniel Goodman]] 17:01, 31 July 2008 (UTC)''':I just put this table together, but I have yet to add the plasmid maps to these. For now, to see the maps, go to [http://bgsc.org BGSC homepage] and find them in the catalog. They are also in the black Turing Group binder under "ordered vectors and strains".  


= Integration Vectors =
= Integration Vectors =
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* 4 pstI sites, EcoRI site, no Xba site
* 4 pstI sites, EcoRI site, no Xba site
| <nowiki>*</nowiki>: can use PyrD & AmyE instead
| <nowiki>*</nowiki>: can use PyrD & AmyE instead
| bgcolor="red" fontcolor="white"| No: did not appear on gel
| bgcolor="red" fontcolor="white"| No: wrong size
|-
|-
| ECE176
| ECE176
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* 2 pstI sites, X and S before promoter, E after terminator
* 2 pstI sites, X and S before promoter, E after terminator
| <nowiki>*</nowiki>: use of strong rpsD promoter, but hard to insert into
| <nowiki>*</nowiki>: use of strong rpsD promoter, but hard to insert into
| bgcolor="red" fontcolor="white"| No: unsure of enzymes/too faint
| bgcolor="green" fontcolor="white"| Yes
|-
|-
| ECE153
| ECE153
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* multiple Xbas, multiple pstIs
* multiple Xbas, multiple pstIs
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence
| bgcolor="yellow" fontcolor="white"| inconclusive
| bgcolor="green" fontcolor="white"| yes
|-
|-
| ECE162
| ECE162
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* multiple Xbas, multiple pstIs
* multiple Xbas, multiple pstIs
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence
| bgcolor="yellow" fontcolor="white"| inconclusive
| bgcolor="yellow" fontcolor="white"| inconclusive : only one band


|-
|-
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| [[image:ECE149.jpg|thumb|ECE149 Map]]
| [[image:ECE149.jpg|thumb|ECE149 Map]]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutingfp.htm From Bayreuth]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutingfp.htm From Bayreuth]
| bgcolor="red" fontcolor="white"| No: no good bands on the gel
| bgcolor="red" fontcolor="white"| No: 3 bands on the gel
|-
|-
| ECE150
| ECE150
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| [[image:ECE153.jpg|thumb|ECE150 Map]]
| [[image:ECE153.jpg|thumb|ECE150 Map]]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincfp.htm From Bayreuth] <br/> (sequence incomplete)
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincfp.htm From Bayreuth] <br/> (sequence incomplete)
| bgcolor="yellow" fontcolor="white"| Inconclusive
| bgcolor="green" fontcolor="white"| yes
|-
|-
| ECE151
| ECE151
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| [[image:ECE147.jpg|thumb|ECE147 Map]]
| [[image:ECE147.jpg|thumb|ECE147 Map]]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincmyc.htm From Bayreuth]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincmyc.htm From Bayreuth]
| bgcolor="yellow" fontcolor="white"| Inconclusive
| bgcolor="green" fontcolor="white"| yes
|}
|}


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* Ampicillin selectable only in EC
* Ampicillin selectable only in EC
* gfpmut3a in front of MCS, no promoter, but gram-positive RBS
* gfpmut3a in front of MCS, no promoter, but gram-positive RBS
| [[image:ECE153.jpg|thumb|ECE165 Map]]
| [[image:ECE165.jpg|thumb|ECE165 Map]]
| [http://www.bgsc.org/sequences/pad123.htm BGSC sequence]
| [http://www.bgsc.org/sequences/pad123.htm BGSC sequence]
| bgcolor="green" fontcolor="white"| Yes
| bgcolor="green" fontcolor="white"| Yes
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| [[image:ECE188.jpg|thumb|ECE190 Map]]
| [[image:ECE188.jpg|thumb|ECE190 Map]]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC05.htm From Bayreuth]
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC05.htm From Bayreuth]
| bgcolor="white" fontcolor="white"| Not yet checked, DNA stocks
| bgcolor="green" fontcolor="white"| Yes
|}
|}


Latest revision as of 04:57, 29 August 2008

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

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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 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
 From Bayreuth Yes


Plasmid Gels

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