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Team:Groningen/Notebook/24 August 2009
From 2009.igem.org
[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, pArsR (4x), pCueO (3x), pZntR (3x)
- → The concentration of plasmids was not determined before restriction was done, and likely the concentration was to high to give a band at ~3000bp of uncut plasmid.
- → From left to right: 1kB ladder, pArsR (2x), pCueO (2x), pZntR (2x), Control BBa_J23101, Empty Slot
- → The 2% Agarose gel was run for 15 min. at 100V to get separation at low bp fragments, larger parts are grouped together at the top
- → The expected sizes are seen in the gel, plasmids can be send for sequencing!
Transporters
Metal Accumulation
- Check pSB1AC3-mymT, fmt, smtA
- Pick 3 colonies of E. coli TOP10 + pSB1AC3-mymT, fmt, smtA.
- Add 1uL MQ and microwave 1min to lyse the cells.
- Do colony PCR with VR/VF primers and RBS-fw/gene specific rev primers.
- Run PCR products on gel.
- From left to right:
- Upper: SmtA-1 till 3 (1200bp), fMT 4-6 (500bp), MymT 7-8(500), AC3 (315bp) (using VF2/VR primers)
- Lower: SmtA-1 till 3 (900bp), fMT 4-6 (200bp), MymT 7-9(215bp) (using gene specific primers)
- Thereby the construct of Mymt-pSB1AC3 nr7-9 do not give correct fragment size, also the constructs pSB1AC3-SmtA nr 1 and pSB1AC3-fMT nr 5 do not give correct fragment size.
- Put o/n culture @ warm room of pSB1AC3-SmtA nr2&3 and pSB1AC3-fMT nr 4&6.
- Cloning of MymT into pSB1AC3 or other vectors has stopped
- It is considered to be to much time consuming to also clone MymT next to the other MTs.
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.
- pMB1* and ColE1 are in different [http://wolfson.huji.ac.il/expression/vector/origin.html#comp compatibility group]
- pMB1 and ColE1 are too similar and thus not compatible
* pMB1 is the pUC19 derived ori present in pSB1AC3
Dry
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