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Team:Groningen/Notebook/1 September 2009
From 2009.igem.org
(Difference between revisions)
m (New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== ===Transporters=== ===Metal Accumulation=== ====MBP-ArsR==== *{{todo|Check sequence}} *{{todo|Put promotors in front}} ...) |
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===GVP Cluster=== | ===GVP Cluster=== | ||
| + | |||
| + | :→ {{todo}} Isolate plasmids from o.n. cultures | ||
| + | :→ {{todo}} Determine concentrations (and prepare for ligation of GVP behind LAC/pBAD promoters) | ||
| + | |||
| + | :→ {{todo}} Make glycerol stocks of pZntR+RBS, pArsR+RBS, pCueO+RBS, pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid) | ||
| + | |||
| + | :→ {{todo}} Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3 | ||
| + | |||
| + | :→ {{todo}} Transform E.coli TOP10 cells with [http://partsregistry.org/Part:pSB2K3 pSB2K3] plasmid from the registry (plate 1, 7C with RFP Coding Device [http://partsregistry.org/Part:BBa_J04450 BBa_J04450]) | ||
| + | |||
| + | :→ {{todo}} Test control of bouyancy in Saline solution | ||
| + | :→ {{todo}} Order synthetic DNA for GVP | ||
| + | :→ {{todo}} Order primer for PstI site removal | ||
| + | |||
| + | :→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | ||
| + | :→ {{todo}} Enter sequences of constructs to Sandbox | ||
| + | |||
| + | |||
| + | '''Overnight Precultures''' | ||
| + | |||
| + | All tubes showed growth of E.coli cells, and must have ampicillin resistance on a plasmid(The tubes differed in cell density though, the ones with 10mL LB were less dense as expected). To be sure of correct ligation products in the plasmids an isolation will be performed, followed by restriction analysis. | ||
| + | |||
| + | |||
| + | '''Overnight Plates''' | ||
| + | |||
| + | The plates grown overnight showed single colony growth, the plates were stored in the fridge for preculture inoculation. | ||
| + | |||
| + | |||
| + | '''Transformation''' | ||
| + | |||
| + | The [http://partsregistry.org/Part:pSB2K3 pSB2K3] vector was chosen to use together with the [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] vector to create a two plasmid system. The ME<sup>tal</sup> promoters with GVP on pSB2K3, and accumulation with transport proteins on pSB1AC3. These two vectors should be compatible with respect to their origins of replication. | ||
| + | |||
| + | |||
| + | [[Image:PSB2K3.jpg|400px]] [[Image:PSB1AC3.jpg|400px]] | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 07:39, 1 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → TODO Isolate plasmids from o.n. cultures
- → TODO Determine concentrations (and prepare for ligation of GVP behind LAC/pBAD promoters)
- → TODO Make glycerol stocks of pZntR+RBS, pArsR+RBS, pCueO+RBS, pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid)
- → TODO Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3
- → TODO Transform E.coli TOP10 cells with [http://partsregistry.org/Part:pSB2K3 pSB2K3] plasmid from the registry (plate 1, 7C with RFP Coding Device [http://partsregistry.org/Part:BBa_J04450 BBa_J04450])
- → TODO Test control of bouyancy in Saline solution
- → TODO Order synthetic DNA for GVP
- → TODO Order primer for PstI site removal
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Overnight Precultures
All tubes showed growth of E.coli cells, and must have ampicillin resistance on a plasmid(The tubes differed in cell density though, the ones with 10mL LB were less dense as expected). To be sure of correct ligation products in the plasmids an isolation will be performed, followed by restriction analysis.
Overnight Plates
The plates grown overnight showed single colony growth, the plates were stored in the fridge for preculture inoculation.
Transformation
The [http://partsregistry.org/Part:pSB2K3 pSB2K3] vector was chosen to use together with the [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] vector to create a two plasmid system. The MEtal promoters with GVP on pSB2K3, and accumulation with transport proteins on pSB1AC3. These two vectors should be compatible with respect to their origins of replication.
Transporters
Metal Accumulation
MBP-ArsR
- Check sequence
- Put promotors in front
- Put transporters in back
fMT
- Plasmid isolation & Restriction analysis
- Put promotors in front
SmtA
- Plasmid isolation & Restriction analysis
- Put promotors in front
Vectors
Dry
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