Part:BBa_K4390024
CBD-tagged Metallothionein (CBD-MT)
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 1 part formed by assembly of the following level 0 parts:
| Promoter | J23100 |
| RBS | B0034 |
| N part | K4390016 |
| O part | K4390023 |
| C part | K4390009 |
| Terminator | K4390001 |
Usage and Biology
Metallothionein (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 cellulose-binding domain (CBD) is the non-catalytic domain of cellulase that recognises the beta-1,4-glycosidic linkage of cellulose and cellulose-derived polymers (e.g. carboxymethylcellulose). In the design, we used CenA for our CBD, the cellulose-binding domain of the cellulase endoglucanase A from Cellulomonas fimi (Din, N. et al., 1994). We attached the CBD to the N-terminus of our MTs via a glycine-serine linker. The CBD binds to the beta-1,4 glycosidic bonds of cellulose and its derivatives like CMC (Din, N. et al., 1994), allowing attachment of the fused MT to the hydrogel matrix.
This part is an expression construct for a metallothionein to bind a Cellulose Hydrogel. It encodes the metallothionein of Mytilus edulis fused to the CBD from the cellulase beta-1,4-endoglucanase of Cellulomonas fimi. CBD tagging would immobilise metallothionein to our carboxymethylcellulose-based hydrogels, this would complete the construction of our 3C hydrogels for sequestration of heavy metal ions from the water. A 6xHis tag and TEV protease cleavage site was also added on the N terminus, downstream with a Cellulose Binding Domain and Gly-Ser linker. The 6xHis tag allows for nickel affinity purification, and the TEV protease cleavage site allows for us to remove the His tag. The Cellulose Binding domain binds to cellulose in our hydrogel, and the Gly-Ser linker ensures enough space is between the Cellulose Binding Domain and metallothionein.
We expressed CBD-tagged M. edulis MTs in the non-pathogenic laboratory E. coli strain, BL21(DE3). Afterwards, we attached our recombinant MTs to our hydrogels by incubating the hydrogels in the lysates of the E. coli cultures expressing the CBD-tagged MTs.
Characterization
After protein expression in the BL21(DE3) cell cultures, the cultures were lysed by sonication, and the lysates were run on an SDS-PAGE gel to confirm the presence of our CBD fused proteins (Figure 1). This SDS-PAGE step also serves as a solubility test to confirm that all of our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods.
Figure 2. SDS-PAGE gel of the lysates containing our expressed constructs. Lane 0 represents the negative control, which is the BL21(DE3) strain containing only the pJUMP29 LacZ acceptor plasmid without any insert. The red lines indicate the bands representing our constructs. The ladder we used was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S).
Result and discussion
From SDS-PAGE, the CBD fused proteins was successfully expressed in BL21(DE3). Then the cells were lysed by sonication and lysate was flowed through hydrogel to allow CBD binding. After we obtain complete MT hydrogel, the hydrogel was put into solution containing Zn and Ni ions. The initial and final metal ion concentrations in the supernatants were measured by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS measures the concentration of a certain metal ion in solution. Therefore, the reduction in the number of unbound metal ions in the supernatant represents the number of metal ions sequestered in the 3C hydrogels (Figure 2).
Figure 2. Amounts of (A) Zn (II) and (B) Ni (II) ions captured by 3C hydrogels. The 3C hydrogel fragments were washed with the buffer (0.4 M Tris-HCl, pH 7.5) 3 times, then incubated in 1 ml of 100 uM Zn (II) or Ni (II) solution (using the buffer as the solvent). The values were obtained by multiplying the molecular weight of the metal ion with the difference between the initial and final concentrations of metal ion in the supernatant, which were measured using ICP-MS. Because of varying weights of the hydrogel fragments, the quantity of metal ion sequestered was divided by the weight of the hydrogel fragment used and multiplied by 20 to calculate the data representative of a 20 mg hydrogel fragment.
Sadly we saw no significant difference between the control and the 3C hydrogel decorated with CBD-tagged metallothionein. The most probable reason for this would be that the hydrogel matrix plays a major role in metal ion sequestration, as we saw this effect in earlier experiments. Another reason may be that the CBD tag is affecting the metallothionein folding and therefore reducing its metal binding capacity, we also saw this effect in earlier results.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
Din, N., Forsythe, I., Burtnick, L., Gilkes, N., Miller, R., Warren, R. and Kilburn, D. (1994). The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding. Molecular Microbiology, 11(4), pp.747-755.
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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