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. | |||
= Integration Vectors = | = Integration Vectors = | ||
Line 23: | Line 22: | ||
! Vector Map | ! Vector Map | ||
! Vector Sequence | ! Vector Sequence | ||
! Possible Uses | |||
! Necessity (1-5 stars) | |||
! Checked on Gel | |||
|- | |- | ||
| ECE112 | | ECE112 | ||
Line 33: | Line 35: | ||
* cassette insertion | * cassette insertion | ||
| amyE | | amyE | ||
| | | [[image:ECE112.jpg|thumb|ECE112 Map]] | ||
| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=1185558 U46196] | | [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=1185558 U46196] | ||
| | |||
* Quantitative testing promoters using Beta-galactosidase assay | |||
* Biobricking: Has multiple pstI sites, Xba is not in cassette | |||
| <nowiki>****</nowiki> | |||
| bgcolor="green" fontcolor="white" | Yes | |||
|- | |- | ||
| ECE171 | | ECE171 | ||
Line 43: | Line 50: | ||
* cassette insertion | * cassette insertion | ||
| pyrD | | pyrD | ||
| | | [[image:ECE171.jpg|thumb|ECE171 Map]] | ||
| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726213 AY464559] | | [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726213 AY464559] | ||
| | |||
* Inserting genes at neutral site | |||
* Can be used for biobrick insertion, all sites within cassette, PstI site is not at MCS | |||
| <nowiki>****</nowiki> | |||
| bgcolor="green" fontcolor="white" | Yes | |||
|- | |- | ||
| ECE172 | | ECE172 | ||
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* cassette insertion | * cassette insertion | ||
| gltA | | gltA | ||
| | | [[image:ECE172.jpg|thumb|ECE172 Map]] | ||
| [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726216 AY464560] | | [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=39726216 AY464560] | ||
| | |||
* Inserting genes at neutral site | |||
* 4 pstI sites, EcoRI site, no Xba site | |||
| <nowiki>*</nowiki>: can use PyrD & AmyE instead | |||
| bgcolor="red" fontcolor="white"| No: wrong size | |||
|- | |- | ||
| ECE176 | | ECE176 | ||
Line 64: | Line 81: | ||
* contains strong rspD promoter and RBS | * contains strong rspD promoter and RBS | ||
| rspD | | rspD | ||
| | | [[image:ECE176.jpg|thumb|ECE176 Map]] | ||
| [http://www.bgsc.org/sequences/pBCJ164.htm BGSC sequence] | | [http://www.bgsc.org/sequences/pBCJ164.htm BGSC sequence] | ||
| | |||
* Inserting genes at neutral site | |||
* Using strong rspD promoter | |||
* 2 pstI sites, X and S before promoter, E after terminator | |||
| <nowiki>*</nowiki>: use of strong rpsD promoter, but hard to insert into | |||
| bgcolor="green" fontcolor="white"| Yes | |||
|- | |- | ||
| ECE153 | | ECE153 | ||
Line 76: | Line 99: | ||
* high fluorescence GFP variant for protein fusions | * high fluorescence GFP variant for protein fusions | ||
| amyE | | amyE | ||
| | | [[image:ECE153.jpg|thumb|ECE153 Map]] | ||
| not available | | not available | ||
| | |||
* GFP-fusion tracking of proteins (for agr components?) | |||
* multiple Xbas, multiple pstIs | |||
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence | |||
| bgcolor="green" fontcolor="white"| yes | |||
|- | |- | ||
| ECE162 | | ECE162 | ||
Line 88: | Line 116: | ||
* cassette insertion | * cassette insertion | ||
| amyE | | amyE | ||
| | | [[image:ECE162.jpg|thumb|ECE162 Map]] | ||
| not available | | not available | ||
| | |||
* GFP-fusion tracking of proteins (for agr components?) | |||
* multiple Xbas, multiple pstIs | |||
| <nowiki>***</nowiki>: inducible promoter, strong gfp, no sequence | |||
| bgcolor="yellow" fontcolor="white"| inconclusive : only one band | |||
|- | |- | ||
|} | |} | ||
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! Vector Map | ! Vector Map | ||
! Vector Sequence | ! Vector Sequence | ||
! Checked on gel | |||
|- | |- | ||
| ECE149 | | ECE149 | ||
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* IPTG-inducible | * IPTG-inducible | ||
* full plasmid insertion | * full plasmid insertion | ||
| | | [[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: 3 bands on the gel | |||
|- | |- | ||
| ECE150 | | ECE150 | ||
Line 123: | Line 159: | ||
* IPTG-inducible | * IPTG-inducible | ||
* full plasmid insertion | * full plasmid insertion | ||
| | | [[image:ECE153.jpg|thumb|ECE150 Map]] | ||
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincfp.htm From Bayreuth] | | [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutincfp.htm From Bayreuth] <br/> (sequence incomplete) | ||
| bgcolor="green" fontcolor="white"| yes | |||
|- | |- | ||
| ECE151 | | ECE151 | ||
Line 134: | Line 171: | ||
* IPTG-inducible | * IPTG-inducible | ||
* full plasmid insertion | * full plasmid insertion | ||
| | | [[image:ECE151.jpg|thumb|ECE151 Map]] | ||
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutinyfp.htm From Bayreuth] | | [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pmutinyfp.htm From Bayreuth] | ||
| bgcolor="green" fontcolor="white"| Yes | |||
|- | |- | ||
| ECE147 | | ECE147 | ||
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* cMyc antibody tag fusion | * cMyc antibody tag fusion | ||
* IPTG-inducible Pspac promoter | * IPTG-inducible Pspac promoter | ||
| | | [[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="green" fontcolor="white"| yes | |||
|} | |} | ||
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! Vector Map | ! Vector Map | ||
! Vector Sequence | ! Vector Sequence | ||
! Checked on gel | |||
|- | |- | ||
| ECE165 | | ECE165 | ||
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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: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 | |||
|- | |- | ||
| ECE166 | | ECE166 | ||
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* Ampicillin selectable only in EC | * Ampicillin selectable only in EC | ||
* gfpmut3a in front of MCS, strong Pupp promoter, gram-positive RBS | * gfpmut3a in front of MCS, strong Pupp promoter, gram-positive RBS | ||
| | | [[image:ECE166.jpg|thumb|ECE166 Map]] | ||
| [http://www.bgsc.org/sequences/pad43-25.htm BGSC sequence ] | | [http://www.bgsc.org/sequences/pad43-25.htm BGSC sequence ] | ||
| bgcolor="green" fontcolor="white"| Yes | |||
|- | |- | ||
| ECE188 | | ECE188 | ||
Line 182: | Line 224: | ||
* Ampicillin selectable only in EC | * Ampicillin selectable only in EC | ||
* weak constitutive PlepA promoter in front of MCS | * weak constitutive PlepA promoter in front of MCS | ||
| | | [[image:ECE188.jpg|thumb|ECE188 Map]] | ||
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC02.htm From Bayreuth] | | [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC02.htm From Bayreuth] | ||
| bgcolor="white" fontcolor="white"| Not yet checked, DNA stocks | |||
|- | |- | ||
| ECE189 | | ECE189 | ||
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* Ampicillin selectable only in EC | * Ampicillin selectable only in EC | ||
* strong xylose inducible PxylA promoter in front of MCS | * strong xylose inducible PxylA promoter in front of MCS | ||
| | | [[image:ECE188.jpg|thumb|ECE189 Map]] | ||
| [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04.htm From Bayreuth] | | [http://www.genetik.uni-bayreuth.de/LSGenetik1/schumann_pHCMC04.htm From Bayreuth] | ||
| bgcolor="white" fontcolor="white"| Not yet checked, DNA stocks | |||
|- | |- | ||
| ECE190 | | ECE190 | ||
Line 200: | Line 244: | ||
* Ampicillin selectable only in EC | * Ampicillin selectable only in EC | ||
* strong xylose inducible PxylA promoter in front of MCS | * strong xylose inducible PxylA promoter in front of MCS | ||
| | | [[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="green" fontcolor="white"| Yes | |||
|} | |} | ||
== [[IGEM:Cambridge/2008/Notebook/Turing_Pattern_Formation/2008/08/04|Plasmid Gels]] == | |||
* We ran these plasmids on gels, using double and single digests, to check them |
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 |
|
amyE | U46196 |
|
**** | Yes | |
ECE171 | pPyr-Kan |
|
pyrD | AY464559 |
|
**** | Yes | |
ECE172 | pGlt-Cm |
|
gltA | AY464560 |
|
*: can use PyrD & AmyE instead | No: wrong size | |
ECE176 | pBCJ164 |
|
rspD | BGSC sequence |
|
*: use of strong rpsD promoter, but hard to insert into | Yes | |
ECE153 | pSG1154 |
|
amyE | not available |
|
***: inducible promoter, strong gfp, no sequence | yes | |
ECE162 | pSG1193 |
|
amyE | not available |
|
***: 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+ |
|
From Bayreuth | No: 3 bands on the gel | |
ECE150 | pMUTIN-CFP |
|
From Bayreuth (sequence incomplete) |
yes | |
ECE151 | pMUTIN-YFP |
|
From Bayreuth | Yes | |
ECE147 | pMUTIN-cMyc |
|
From Bayreuth | yes |
Shuttle Vectors
BGSC Accession | Vector Name | Features | Vector Map | Vector Sequence | Checked on gel |
---|---|---|---|---|---|
ECE165 | pAD123 |
|
BGSC sequence | Yes | |
ECE166 | pAD43-25 |
|
BGSC sequence | Yes | |
ECE188 | pHCMC02 |
|
From Bayreuth | Not yet checked, DNA stocks | |
ECE189 | pHCMC04 |
|
From Bayreuth | Not yet checked, DNA stocks | |
ECE190 | pHCMC05 |
|
From Bayreuth | Yes |
Plasmid Gels
- We ran these plasmids on gels, using double and single digests, to check them