Difference between revisions of "Part:BBa K190019"

Revision as of 21:36, 21 October 2009

fMT

[http://2009.igem.org/Team:Groningen/Project/Accumulation#fMT fMT] is a metallothionein, binding Arsenite(III) and Arsenate(V), it has higher affinity for As(III). The protein was isolated from [http://en.wikipedia.org/wiki/Fucus_vesiculosus Fucus vesiculosus] and described by Morris et al. It consists of 67 amino acid residues and has 16 cysteine residues, a high cysteine content is a key feature of MT. Another characteristic is the lack of aromatic residues is also seen in fMT where it only has one, tryptophan. Two domains containing cysteine residues are presumed to be involved in the metal binding function. Unusual in fMT is the presence of a 14 amino acid linker region between the two putative metal-binding domains which contains no cysteine residues. Being a MT fMT binds a multitude of metal ions, 6 Cd²⁺ ions or 5 As³⁺ ions in a sequential order, facilitated by the elongated linker domain (Ngu 2009).

Usage and Biology

Overexpression of fMT can be used to accumulate Arsenic without toxification of the cells. The gene was functionally expressed in E. coli.

From literature it is known that metallothioneins are degraded inside lysozymes, especially when they are in the apo- (non-bound) state (Gold 2008), for bacteria this is not known, but from mammalian MT this can be estimated up to 0.8nmol apo-MT/mg protein/min (Klaassen 1994). This can be avoided by adding ~0.5mM metal-salts (ZnCl₂, CuSo₄, CdCl₂) to cells expressing the protein. But afterwards the metals should be chelated from the metallothioneins by for instance EDTA.

Sequence and Features

Results

Arsenite uptake by E. coli containing pSB1A2-R0010-fMT (BBa_K190032) was tested using a arsenic uptake assay (according to Kostal 2004) and arsenic concentration determination by ICP-MS (see [http://2009.igem.org/Team:Groningen/Protocols#Metal_uptake_assay_for_E._coliKostal_2004 protocols]). But because of a lack of reproducibility and a unexpected high uptake yield (as can be seen in figure 1), the functionality of fMT could not be determined. The curve of arsenic uptake in E. coli with fMT shows a lower uptake yield than the other strains, this may be caused by an accidental increase of temperature during drying the cells. For further reading on discussion of this data see the [http://2009.igem.org/Team:Groningen/Accumulation wiki]

Figure 1: Uptake of As(III) by E. coli WT, and the strains containing the different parts of the accumulation device. As a control the arsenic uptake of E. coli with ArsR overexpression (as described by Kostal 2004) is also shown.