Part:BBa_K4390019
Callinectes sapidus 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 | K4390011 |
| 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. In this construct, the MT sequence came from Callinectes sapidus, an aquatic crustacean living in high heavy metal rich environments (De Martinez Gaspar Martins and Bianchini, 2009) which induces more cysteine and increased the number of heavy metal ions bound to each MT (Li, X. et al., 2021). 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, Callinectes sapidus MT was compared with MT from Mytilus edulis,Mytilus galloprovincialis, 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 the protein, 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 Callinectes sapidus MT using PS1 and PS2 as primers. The 1 kb ladder (left) and colony PCR products (right) was running through an electrophresis gel to determine the molecular weight of assembled plasmid.
Result
Due to shortage of resources, Callinectes sapidus MT had sadly been dropped from protein expressing part. Whereas from the result of colony PCR, the sequence should be able to express Callinectes sapidus MT in BL21(DE3).
Docking simulation
Non-designed Callinectes sapidus 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 4).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 5 Ag+ docked with Gibbs free energy per ion binding of -0.170 kcal/mol. This data was compared with Mytilus edulis, Mytilus galloprovincialis, Danio rerio, Pseudomonas fluorescens and Saccharomyces cerevisiae
- Figure 2. 3D structure of wilt-type Callinectes sapidus 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
De Martinez Gaspar Martins, C. and Bianchini, A., 2009. Metallothionein-like proteins in the blue crab Callinectes sapidus: Effect of water salinity and ions. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 152(3), pp.366-371.
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.
| None |