Coding

Part:BBa_K4390034

Designed by: Maarten van den Ancker, Serafina Soehianto   Group: iGEM22_Edinburgh-UHAS_Ghana   (2022-08-16)


SpyTag flanked M. edulis Metallothionein

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.

This is a Level 0 part of type O-C part generating the following 4 base overhangs at upstream (AGCC) and downstream (GCTT) ends.

Usage and Biology

SpyTag is a 13 amino acid-long protein that spontaneously reacts with the SpyCatcher protein (12.3 kDa). Together they form an intermolecular isopeptide bond. This provides an alternative to His-tag purification techniques. Insertion of either the SpyTag or the SpyCatcher sequence into a recombinant protein will allow its purification.

etallothionein (MT) is a small protein (around 6-7 kDa) which is rich in cysteine. These thiol group in cysteines provide ability to chelate almost all heavy metal ions including Cd2+, Hg2+, Pb2+ and As3+, but had been shown that has higher binding affinity with Hg2+ (Manceau, A. et al., 2019). The ability of chelating heavy metals provides the metal tolerance for its hosts. This MT sequence was obtained from Mytilus edulis, a blue mussel which originally live in aqueous environment (MACKAY E. A. et al.,1993).

The SpyTag-SpyCatcher protein pair is a modular assembly system. SpyTag and SpyCatcher spontaneously form a covalent interaction with each other. There are Glycine-Serine linkers between the Metallothioneins and SpyTag. This part is used to form SpyTag-SpyCatcher hydrogels, and this part immobilizes Metallothioneins in the hydrogel.

SpyTag-MT-SpyTag is designed to be a cross linker between triple SpyCatcher subunits for hydrogel formation, with metallothionein (MT) acting as the spacer protein between two SpyTags. This also allows covalent immobilisation of MTs to the SpyTag-SpyCatcher hydrogel.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 111
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 111
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 111
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 111
    Illegal NgoMIV site found at 60
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

MACKAY, E. A. et al. (1993) Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis. European journal of biochemistry. 218 (1), 183–194.

Manceau, A. et al. (2019) Mercury(II) Binding to Metallothionein in Mytilus edulis revealed by High Energy‐Resolution XANES Spectroscopy. Chemistry : a European journal. 25 (4), 997–1009.


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Parameters
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