From 2009.igem.org
Wet
GVP Cluster
Over Night Cultures
Over night cultures in 4mL LB-amp100 or LB-kan50 medium were prepared from the following glycerol stocks:
- → E.coli TOP10 pSB3K3-high const. promoter (kan.)
- → E.coli TOP10 pSB3K3-med. const. promoter (kan.)
- → E.coli TOP10 pSB3K3-low const. promoter (kan.)
- → E.coli TOP10 pSB1AC3-high const. promoter (amp.)
- → E.coli TOP10 pSB1AC3-med. const. promoter (amp.)
- → E.coli TOP10 pSB1AC3-low const. promoter (amp.)
and put in the 37°C waterbath at 200 rpm.
Transporters
DONE
GlpF
→ pick colonies and use for overnight culture. (2 colonies in 6 mL LB-amp medium)
Metal Accumulation
TODO
MBP
- Plasmid isolation (cells can be found in the freezer Jolanda)
- PCR of MBP
-
12,5 uL
| Phusion 2x MM
|
1 uL
| MBP Fw
|
1 uL
| MBP rev
|
0.5uL
| MBP plasmid
|
Vectors
Dry
We (Jasper and Klaas) worked on deriving a method for estimating K and Vmax values based on combined import/export measurements. So far the application of this method on figure 3A of Kostal2004 has only yielded nonsensical results (K and V both negative and K (close to) zero if they are constrained to be positive).