Team:Groningen/Notebook/13 August 2009

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Wet

GVP Cluster

DONE inocculate o.n. cultures for glycerol stocks
DONE inocculate o.n. cultures for J61002-H/M/L promoter plasmid isolation
DONE ligation of GVP into vectors
DONE transformation of E.coli TOP10 with ligation product

Bouyancy Test

To test the effect of the metals Cu, Zn and Ar on bouyancy cells containing the metal sensitive promoters + GVP (msGVP) construct are grown in liguid media containing a range of different metal concentrations. As controls there is a reaction tube without Cu, Zn or Ar and reaction tubes with cells that contain the pSB1AC3 vector instead of msGVP.

Procedure:

  • Grow cells o.n. in liquid culture (LB, Amp 100 ug/ml) containing the correct amount of inducer (see table metal concentrations).
  • Centrifuge: 1000rpm, ~20 min
  • Remove medium and resuspend in saline (0.15 mM NaCl solution)
  • The tubes are then kept at room temp. without shaking


Metal Concentrations

Cu 0 0.01 mM 0.1 mM 0.5 mM 1 mM 2 mM
Zn 0 2 uM 4 uM 8 uM 10 uM 15 uM
Ar 0 0.01 mM 0.1 mM ? ? ?


Ligation

(1:3)

  • 4 uL Ligase buffer
  • 1 ul T4 Ligase
  • 9 uL plasmid pSB1AC3-H digested with PstI and SpeI
  • 9 uL insert GVP restricted with XbaI and PstI

(1:3)(2x)

  • 4 uL Ligase buffer
  • 1 ul T4 Ligase
  • 12 uL plasmid pSB3K3-H/L digested with PstI and SpeI
  • 5 uL insert GVP restricted with XbaI and PstI

Incubate:

  • 25°C 60min.
  • kept on ice for 10min.

Tranformation

  • add 10uL of the pSB1AC3-H-GVP and pSB3K3-H/L-GVP ligation products to 50uL competent E.coli TOP10 cells.

Incubate:

  • 30 min @ ice
  • 90 sec 42°C
  • 2 min @ ice
  • add 800uL LB-medium
  • incubate for 1 h at 37°C
  • concentrate in 100uL LB-medium
  • plate on LB-amp50 plates

Restriction Control

Gel Mic 13-8.pdf Generulers 1kb marker Fermentas.jpg

→ From left to right:1kB ladder,

Transporters

Metal Accumulation

  • MymT plus RBS and pre
    • A PCR was done yesterday with HmtA_F1_PR-RBS-L-ATG and MymT rev to amplify MymT with RBS and pre/suff.
    • The PCR product was ran on an 1.5% Agarose gel and the band ~213bp was cut out.
    • Stored in fridge

F102471 2009-08-13 MymT RBS.JPG

from left to right: Marker 1kb, PCR product 1 (2x), Marker 1kb, PCR product 2(2x)
    • The lower band was excised

Vectors

Dry

April
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July
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August
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September
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October
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November
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