Part:BBa_K4390055
M. edulis MT expression construct
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 | K4390006 |
| O part | K4390007 |
| 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). This construct is mainly be used to express wild-type M. edulis MT as control.
Result and Dicussion
MT heavy metal binding affinity improvement
Ensuring Mytilus edulis MT was expressed in BL21(DE3), we performed AgNO3 gradient plate test to test the influence of Mytilus edulis MT on BL21(DE3) heavy metal tolerance. AgNO3 was used based on its high efficiency of antibacterial (Yin, I. X. et al., 2020) with low toxicity towards eukaryote. With the presence of Mytilus edulis MT, BL21(DE3) started to grew at higher AgNO3 concentration plates (Figure 3A, 3B), indicating that Mytilus edulis MT did provide heavy metal tolerance to the host bacteria.
Figure 3. AgNO3 gradient plate test for control, wild-type MT and error prone MT. 10 different concentrations were used from 16-30 mg/L for each test. The cell used in the test are A) the control BL21(DE3) with no MT expressed. B) BL21(DE3) cells expressing wild type Mytilus edulis MT. C) BL21(DE3) cells expressing error prone Mytilus edulis MT.
The result of silver tolerance test was compared between Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Danio rerio, Pseudomonas fluorescens and Saccharomyces cerevisiae to identify the MT which binds to the highest number of heavy metal ions. Error prone PCR of each MT was also performed with different concentration of dNTPs to increase the possibility of cysteine mutation. The result of AgNO3 gradient plate test for error prone PCR products show strange trend with no growth between 18-20 mg/L but appear several colonies on 22mg/L (Figure 3C). This was possibly due to direct evolutionary mutation at high AgNO3 concentration environment, but the possibility of experimental error should also be considered.
Docking simulation
Non-designed Mytilus edulis MT sequence was taken from NCBI and the Alphafold structures shown were predicted (Figure 4). These structures were docked to Ag+ using AutoDock 4.2 such that the structures were hydrated and energy minimised while allowing gamma sulphurs on the sidechains of cysteines to form coordinate covalent bonds with the metal ligand (Figure 5).The energy minimisation was done after each ligand was docked. MTs contain many cysteines however each cysteine does not carry the same binding affinity for the ligand. This was accounted for using a pass/fail metric where the passed cysteine had negative Gibbs free energy thus making the binding spontaneous. As result, there were 4 Ag+ docked with Gibbs free energy per ion of -0.208 kcal/mol. This data was compared with Mytilus galloprovincialis, Callinectes sapidus, Danio rerio, Pseudomonas fluorescens and Saccharomyces cerevisiae. Interestingly, Mytilus edulis which contains more cysteine only bind to fewer Ag+ thus have a lower heavy metal binding efficiency and affinity.
- Figure 5. 3D structure of wilt-type Mytilus edulis MT predicted by Alphafold with the metal ion binding been docked by AutoDock 4.2.
- Table 1. In-silico modelled Gibbs free energy based on docking simulation
| Metallothionein | Total cysteines | Number of Ag+ docked | Total binding free energy (kcal/mol) | Gibbs free energy per ion binding (kcal/mol) |
|---|---|---|---|---|
| M. edulis | 20 | 4 | -0.83 | -0.208 |
| M. galloprovincialis | 21 | 5 | -0.85 | -0.170 |
| D. rerio | 20 | 4 | -0.58 | -0.145 |
| C. sapidus | 18 | 5 | -0.65 | -0.130 |
| P. fluorescens | 9 | 6 | -2.44 | -0.407 |
| S. cerevisiae | 12 | 5 | -1.87 | -0.374 |
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
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.
| None |