Team:Groningen/Notebook/22 August 2009

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Latest revision as of 22:41, 22 August 2009

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-pME^tal+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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23101 BBa_J23101] constitutive promoter 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 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