Team:Groningen/Notebook/25 September 2009

From 2009.igem.org

Igemhomelogo.png

Wet

GVP Cluster

Transporters

Metal Accumulation

  • Harvest E.coli cells and As(III) uptake experiment
    • Take OD sample to deterimine OD600.
    • Harvest E.coli WT (pSB1A2 + pLac), E.coli + pArsR-RFP and E.coli + pLac-fMT by

spinning down @ 6000rpm for 20min in Sorvall centrifuge.

    • Wash 2x with TB74S buffer.
    • Resuspend upto OD=10.
    • Dry 1ml sample for 2hrs @ 65°C.
    • Do uptake experiments with the different samples (1gr cells @ 22mg/ml concentration)
      • At As(III) concentrations for 1hr: 0, 10, 20, 50 and 100uM As(III).
      • And at time points with 10uM As(III) at: 0, 10, 20, 40 and (60)min.
      • For fMT only the concentration samples were taken!
    • Spin down, wash with TB74S and resuspend in 10ml ddH2O.
    • Dry for 2days @ 65°C.
The determined dry weight was 5x lower than expected, so the sample which should have contained at least 1gr dry cells only contained ~100-200mg dry cells.

Vectors

Dry

April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30