Team:Groningen/Notebook/28 July 2009

Transporters
Result of GlpF PCR3 done yesterday.



a band of 903 was seen as expected. The bands were cut out of the gel and gel extraction was done using ..kit. The DNA was used to do another PCR.

The result can be seen below. The bands were cut out of the gel and purified. The concentration was measured using nanodrop (17ng/uL) and stored at -20.



Restriction and ligation was done as can be seen in the image below.



Restiction



Metal Accumulation

 * Transform E.coli TOP10 with SmtA
 * Use normal transformation protocol.
 * Transform 2uL pET29a -SmtA and pGEX-3 -SmtA GST to E. coli TOP10.
 * Plate on LBA-Amp and LBA-Kan.

Vectors
Oligo's for metal dependent promotors were annealed, 5' phosphorylated using T4 polynucleotide kinase (PNK) and let to cool to room temperature. Vectors &  were digested using SpeI and EcoRI and put to 1% agarose gel (see images below). Top bands were isolated from gel and purified. The vector (3 μL) and the oligo mix (1 μL) were mixed and heated to 65 °C for 1 minute. After cooling to roomtemperature the ligase buffer (1 μL) and ligase (1 μL) (and 4 μL MilliQ) were added and the ligation was put to 4 °C overnight.  SEE FOR FULL PROTOCOL: Protocol metal dependent promotors
 * Ligate metal sensitive promoters with pSB3K3 and pSB1AC3


 * Isolate pSB1A2-pBAD
 * Used Sigma plasmid isolation kit
 * Eluted in 50uL MQ
 * Determined concentration by Nanodrop.
 * Restriction digest and ligation tomorrow.

Dry
Jasper started reading about SmtA, but so far no luck in finding any quantitative results that can be used for modelling. At least, it seems that way. The main things that can be found that might be useful are the amount of metal bound to SmtA in Shi1992, a comparison of zinc accumulation with and without smt(A) in Turner1995 (not in E. coli) and PMPS-titration/H+-competition curves in Shi1992 and Blindauer2002. However, it is not yet clear if (and how) this data can be used to derive useful parameters for modelling.