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Team:Groningen/Notebook/13 August 2009
From 2009.igem.org
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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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
- → 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
- from left to right: Marker 1kb, PCR product 1 (2x), Marker 1kb, PCR product 2(2x)
- The lower band was excised
Vectors
Dry
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