From 2009.igem.org
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