Coding

Part:BBa_K1478002

Designed by: Fabian Frömling   Group: iGEM14_Hannover   (2014-09-18)

Top metal binding protein. Binds zinc, arsenic, copper and cadmium.

Multiple domain protein. Domains bind zinc, arsenic, copper and cadmium. Sequences derived from metallothioneins and transcription factors of different organisms. Arsenic, zinc and cadmium binding regions are full length metallothioneins. Zinc binding region derived from a transcritpion factor binding domain. Different protein binding regions are separated with glycerin-serine spacers to improve domain folding. Metal binding regions consists of disulfide bonds. For expression in E. coli disulfide bond forming strains are useful (e. g. Origami).

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

For the analysis we grew large scale E. coli cultures with non-lethal heavy metal concentrations in the media. At high optical density the cultures were pelleted. The pellets were dried to calculate the dryweight. The dried pellets were solubilized in HNO3 under high pressure and temperatures. Afterwards, the samples were analysed via ICP-OES. To quantify the effect of our T4MBP, we compared the results of cells expressing T4MBP to cells without this protein. Figures 1 shows the heavy metal binding efficiency difference between wildtype bacteria without added heavy metal and bacteria plus T4MBP and added heavy metal.

Figure 1 shows that through expressed T4MBP about four times more zinc can be bound to bacteria than to the normal wildtype. A difference of binding cadmium of about 3 times more by T4MBP than without can be seen. The bars for copper with added heavy metal differ the least, probably because of the small sample amount and of the copper-toxicity for the bacteria. Therefore, no exact statement for the copper binding effiency is possible.

Fig. 1: Measured heavy metals in dry bacteria pellets of wildtype bacteria (WT) and of bacteria with T4MBP (left) without added heavy metals and (right) with added heavy metals.

[edit]
Categories
//cds
//chassis/eukaryote/athaliana
//chassis/eukaryote/ntabacum
//chassis/prokaryote/rradiobacter
//proteindomain/binding
Parameters
None