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Team:Groningen/Notebook/24 August 2009
From 2009.igem.org
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:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | :→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | ||
:→ {{todo}} Enter sequences of constructs to Sandbox | :→ {{todo}} Enter sequences of constructs to Sandbox | ||
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| + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
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| + | '''Plasmid Purification''' | ||
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| + | Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190016 Zinc+RBS], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190017 Copper+RBS] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190015 Arsenic+RBS] promoters and RFP with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit". | ||
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| + | * From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded. | ||
| + | * Plasmids were eluted with 30μL MQ and stored in the fridge | ||
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| + | '''Restriction Control''' | ||
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| + | Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out promoter-RFP, and create ~2000bp and ~950bp fragments. | ||
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| + | [[Image:24-8 no.1.jpg|400px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
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| + | :→ From left to right: 1kB ladder, | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 14:51, 24 August 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 Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
- → TODO Gel purification of wanted fragments and nanodrop
- → TODO Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
- → TODO Send plasmids with MEtal promoters in BBa_J61002 vector for sequencing
- → TODO Check MEtal + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190016 Zinc+RBS], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190017 Copper+RBS] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190015 Arsenic+RBS] promoters and RFP with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 30μL MQ and stored in the fridge
Restriction Control
Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out promoter-RFP, and create ~2000bp and ~950bp fragments.
- → From left to right: 1kB ladder,
Transporters
Metal Accumulation
Vectors
Metal promotors
[http://wolfson.huji.ac.il/expression/vector/origin.html#top origin of replication] determines:
- Vector copy number
- Plasmid compatibility: its ability to replicate in conjuction with another plasmid.
- Plasmids that utilize the same replication system cannot co-exist in the same bacterial cell.
- pUC19 and ColE1 are in different [http://wolfson.huji.ac.il/expression/vector/origin.html#comp compatibility group]
Dry
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