Part:BBa_K4390015
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 | K4390016 |
| O part | K4390017 |
| 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. For its ability to binding heavy metal strongly, this part can be used to build structure which can capture heavy metal ions in aqueous environment. This MT sequence was obtained from Mytilus edulis, a blue mussel which originally live in aqueous environment (MACKAY E. A. et al.,1993). Mytilus edulis MT was our mainly used protein for heavy metal bioremediation part but was also compared with MT from Mytilus galloprovincialis, Callinectes sapidus, Danio rerio, Pseudomonas fluorescens and Saccharomyces cerevisiae for their ability to chelate more heavy metals which lead to higher heavy metal tolerance in BL21(DE3). To express and purify the protein, the sequence was designed as a C part for JUMP assembly (Valenzuela-Ortega M and French C., 2021).
Characterization
MT heavy metal binding affinity improvement
Mytilus edulis MT part was assembled into plasmid pJUMP29-1A(lacZ) along with SUMO tag as N part and 6-His tag as O part. The SUMO tag is added to stablize the protein in E. coli (Li, X. et al., 2021), the His tag is used to purify the protein after expressing in BL21(DE3) cells. To confirm the assembly was success, we performed blue-white colony screening and colony PCR. The transformed cells were plate on Kanamycin and X-gal plates. Since pJUMP29-1A(lacZ) contains lacZ as a cloning receptor, the beta-galactosidase encoded by lacZ will cleave X-gal and forming a molecule which dimerizes and turns the colony blue when assembly is failed. It is possible that the lacZ in pJUMP29-1A(lacZ) was cut out and the non-complementary sticky ends were annealled by T4 ligase. Therefore we picked up white colonies and performed colony PCR to ensure that the assembly was correct (Figure 1).
Figure 1. Colony PCR of Mytilus edulis MT using PS1 and PS2 as primers. The 1 kb ladder (left) and colony PCR products (right) was running through a electrophresis gel to determine the molecular weight of assembled plasmid.
Hydrogel heavy metal bioremediation
Mytilus edulis MT was assembled into pJUMP29-1A(lacZ) along with 6-His tag as N part and celloluse binding domain as O part. We then transformed them into E. coli BL21(DE3) for protein expression, and produced non-tagged sfGFP to use as controls for the fluorescence assays for assessing the performance of the CBD and SB7 tags. 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- and SB7-tagged proteins (Figure 2).
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 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.
Hydrogel heavy metal bioremediation
After protein expression, 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 4).
Figure 4. 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.
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
Li, X. et al. (2021) Genetic modifications of metallothionein enhance the tolerance and bioaccumulation of heavy metals in Escherichia coli. Ecotoxicology and environmental safety. 222112512–112512.
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.
Valenzuela-Ortega, M. & French, C. (2021) Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology. Synthetic biology (Oxford University Press). 6 (1), ysab003–ysab003.
Yin, I. X. et al. (2020) The Antibacterial Mechanism of Silver Nanoparticles and Its Application in Dentistry. International journal of nanomedicine. 152555–2562.
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