Team:Groningen/Notebook/22 August 2009

From 2009.igem.org

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Wet

GVP Cluster

DONE Isolate plasmids from E.coli Top10 BBa_J23101 cells
DONE Plate E.coli Top10 BBa_J23101 cells from o.n. culture
TODO Pellet remaining cells for short storage in -20 box (not needed)
DONE Place the four test cultures with and without GVP on table at room temperature (make foto)
DONE 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.
www.sigmaaldrich.com

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).

Transporters

Metal Accumulation

  • Transform E. coli with pSB1AC3-fMT, MymT, SmtA ligations
    • Use chemically competent cells and transform using heat shock (37°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°C

Vectors

Dry

April
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