Team:Groningen/Notebook/22 August 2009

GVP Cluster

 * → ✅ Isolate plasmids from E.coli Top10 BBa_J23101 cells
 * → ✅ Plate E.coli Top10 BBa_J23101 cells from o.n. culture
 * → Pellet remaining cells for short storage in -20 box (not needed)


 * → ✅ Place the four test cultures with and without GVP on table at room temperature (make foto)


 * → ✅ Look at plates stored at 37C with BBa_J61002-pArsR+/pZntR+/pCueO+ and use colonies to grow o.n. cultures for plasmid isolation

Plates

Showed single colony growth on plates with J61002-pMEtal+RBS-RFP plasmids, and were stored in the fridge for future preculture growth.


 * → The plates with high and low concentration of transformed cells showed colonies in the expected ratio.


 * → On the plates for pZntR+ and pCueO+ no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.


 * → The plates with pArsR+ plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP.

Over Night Cultures


 * → Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation.



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with BBa_J23101 constitutive promoter with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Plasmids were eluted with 40μL MQ and stored in the fridge

New over night cultures (2 days)

The six plates containing colonies of E.coli TOP10 with BBa_J61002-pArsR+/pZntR+/pCueO+ were used to inoculate four tubes with 5 mL LB-amp100 for each promoter.


 * → Instead of an over night incubation, the incubation time was increased to two nights, because it was sunday.

Buoyancy Test

Four tubes with 6mL LB-amp100 were inoculated with E.coli TOP10 pNL29, pSB1AC3-M/L-GVP, and pSB1AC3-RFP yesterday afternoon. All tubes had cell growth of comparable size, but no OD600 measurement was made.


 * → The tubes were placed in a foam support to let the cells come to rest and placed at room temperature (a picture was taken as time zero, see below).

Metal Accumulation

 * Transform E. coli with pSB1AC3-fMT, MymT, SmtA ligations
 * Use chemically competent cells and transform using heat shock (37&deg;C)
 * Transform ligation mixtures of
 * pSB1AC3 + MymT
 * pSB1AC3 + SmtA
 * pSB1AC3 + fMT
 * pSB1AC3 (self ligation control)
 * pSB1AC3-H + RFP (31-07-09; positive control)
 * MQ (negative control)


 * Plate on LBAmp and put o/n in 37&deg;C