Team:Groningen/Notebook/3 August 2009

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
✅ GlpF &rarr; pick colonies and use for overnight culture. (2 colonies in 6 mL LB-amp medium)

Metal Accumulation
MBP
 * Plasmid isolation (cells can be found in the freezer Jolanda)
 * PCR of MBP
 * PCR Mix


 * Restriction digest of pET-29a + SmtA and pGEX-3 + SmtA-GST 
 * Digest 500ng pET-29a + SmtA and pGEX-3 + SmtA-GST with AvaI(FD) in a total volume of 20uL.
 * Incubate 30min @ 37;deg
 * Run 10uL + 5uL loading dye on an 1% Agarose gel
 * From left to right:
 * 2=1kb marker
 * 4=SmtA-GST nr1/AvaI
 * 5=SmtA-GST nr2/AvaI
 * 6=SmtA-GST nr3/AvaI
 * 8=SmtA nr1/AvaI
 * 9=SmtA nr2/AvaI


 * Expected fragment size:
 * pGEX ~5000, SmtA-GST ~200
 * pET29a ~4000, ~1200 SmtA ~200

The fragments of 200bp cannot be seen on gel, but the fragments of 4000 and 5000 can be seen.. For pGEX-3 + SmtA-GST only the third sample seems to be digested properly. So this construct should be checked again.

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).

we used the data of the plasmid, in the e. coli we used the chromosal ArsR, to see the difrence between the genes I have the data alignment with clustalW