Team:Groningen/Notebook/24 August 2009
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- | :→ From left to right: 1kB ladder, | + | :→ From left to right: 1kB ladder, Ars, Cue, Znt |
===Transporters=== | ===Transporters=== |
Revision as of 14:52, 24 August 2009
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 BBa_J61002 with Zinc+RBS, Copper+RBS and Arsenic+RBS promoters and RFP with the "Sygma-Aldrich™ 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, Ars, Cue, Znt
Transporters
Metal Accumulation
Vectors
Metal promotors
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 compatibility group
Dry
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