Team:Groningen/Notebook/13 August 2009

GVP Cluster

 * → ✅ inocculate o.n. cultures for glycerol stocks
 * → ✅ inocculate o.n. cultures for J61002-H/M/L promoter plasmid isolation
 * → ✅ ligation of GVP into vectors
 * → ✅ 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

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




 * → From left to right:1kB ladder,

HmtA
Restriction check on ON culture of picked colony's from the transformants plate of the ligation of pSB1AC3+HmtA both cut with EcoRI and SpeI. HmtA still contains PstI sites. Restriction checked with PstI that should result in 3 fragments:4136bp 702bp 237bp shown in lane 4,5,6 and 8. those plasmids were transformed an plated on LB-Amp plates as working stocks.

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
 * from left to right: Marker 1kb, PCR product 1 (2x), Marker 1kb, PCR product 2(2x)


 * The lower band was excised, why there is an upper band is unknown.