Team:Groningen/Notebook/13 August 2009

From 2009.igem.org

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

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,

Transporters

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.

Vectors

Dry