Difference between revisions of "Part:BBa K4390009"

Latest revision as of 16:12, 8 October 2022

Mytilus edulis Metallothionein

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 0 part of type C part generating the following 4 base overhangs at upstream (TTCG) and downstream (GCTT) ends.

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 is required to be assembled into plasmid pJUMP29-1A(lacZ) along with BBa_K4390017 and BBa_K4390016. To confirm the assembly was success, we performed blue-white colony screening and colony PCR. 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. The transformed cells were plate on Kanamycin and X-gal plates with blue-white screen and colony PCR used to test the assembly (Figure 2).

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) 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 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 most 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 error prone PCR 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

To be added

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 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 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 4. 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

Sequence and Features

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.

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.