Part:BBa_K4390020
Danio rerio 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 | K4390012 |
| 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 Danio rerio, a well-characterised model organism which makes its exploration relevant to ongoing research (Chan et al., 2006; Butt and Ecker, 1987). This MT is also an isoform which contains higher cysteine frequency with 23 in 72 amino acids (Vergani, L. et al., 2007). The SUMO tag is added to stablize the protein in E. coli (Li, X. et al., 2021) and the 6-His tag is used to purify the protein after expressing in BL21(DE3) cells. To improve the heavy metal binding affinity, Danio rerio MT was compared with MT from Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, 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 the protein in BL21(DE3), this construction was assembled into plasmid pJUMP29-1A(lacZ).
Characterization
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 Danio rerio 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.
Result and discussion
MT heavy metal binding affinity improvement
Ensuring Danio rerio MT was expressed in BL21(DE3), we performed AgNO3 gradient plate test to test the influence of Mytilus galloprovincialis 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 Danio rerio MT, BL21(DE3) started to grew at higher AgNO3 concentration plates (Figure 2A, 2B), indicating that Danio rerio MT did provide heavy metal tolerance to the host bacteria.
Figure 2. AgNO3 gradient plate test for control, wild-type 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 Danio rerio MT. C) BL21(DE3) cells expressing error prone Danio rerio MT.
The result of silver tolerance test was compared between MTs from Danio rerio, Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, 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 one colony grew at a relatively higher AgNO3 concentration (26 mg/L) which shown almost no growth for other type of MTs (Figure 2C). This was possibly due to mutation on MT which increased the heavy metal binding affinity, but the possibility of experimental error should also be considered.
Docking simulation
Non-designed Danio rerio MT sequence was taken from NCBI and the Alphafold structures shown were predicted (Figure 3). 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 3).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 -0.145 kcal/mol. This data was compared with Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Pseudomonas fluorescens and Saccharomyces cerevisiae With the same number of cysteine, Danio rerio MT and Mytilus edulis MT Ag+ binding affinity are similar.
- Figure 3. 3D structure of wilt-type Danio rerio 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
Chan, K. M. et al. (2006) Metallothionein gene expression in zebrafish embryo-larvae and ZFL cell-line exposed to heavy metal ions. Marine environmental research. 62S83–S87.
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.
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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