Team:Groningen/Notebook/28 July 2009

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Wet

GVP Cluster

Transporters

Result of GlpF PCR3 done yesterday.

GlpFPCR3 noted.JPG

a band of 903 was seen as expected. The bands were cut out of the gel and gel extraction was done using ..kit.

Metal Accumulation

Vectors

Oligo's for metal dependent promotors were annealed, 5' phosphorylated using T4 polynucleotide kinase (PNK) and let to cool to room temperature. Vectors pSB3K3 & pSB1AC3 were digested using SpeI and EcoRI and put to 1% agarose gel (see images). 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. PSB1AC3 restricted with EcoRi & SpeI - 28july2009.JPG PSB3K3 restricted with EcoRI & SpeI - 28july2009.JPG SEE FOR FULL PROTOCOL: Protocol metal dependent promotors

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.


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