Team:Groningen/Notebook/24 August 2009

GVP Cluster

 * → Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
 * → Gel purification of wanted fragments and nanodrop
 * → Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
 * → Send plasmids with MEtal promoters in BBa_J61002 vector for sequencing
 * → Check MEtal + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)


 * → Test promoter strenght compared to BBa_J23101 promoter (Sven)
 * → 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, 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!

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.

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.
 * pMB1* and ColE1 are in different compatibility group
 * pMB1 and ColE1 are too similar and thus not compatible
 * pMB1 is the pUC19 derived ori present in pSB1AC3