Part:BBa_K4390021
Pseudomonas fluorescens 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 | K4390013 |
| Terminator | K4390001 |
Usage and Biology
Metallothionein (MT) is a small protein (around 6-7 kDa) which is cysteine rich. 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 came from Pseudomonas fluorescens because the MT was studied to account for potential differences in prokaryote and eukaryote (Habjanič et al., 2020; Olafson, 1984). 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, Pseudomonas fluorescens MT was designed to compared with MT from Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Danio rerio 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 also designed as a C part for JUMP assembly (Valenzuela-Ortega M and French C., 2021). But due to the designing mistake, this MT was not able to be assembled and expressed.
Characterization
To check whether the assembly was success, we performed blue-white colony screening. There were only few white colonies appear on the plate, indicating that the assembly might not work successfully. To test whether those white colonies contain correct assemblies, colony PCR was performed with no correct bands appear. After general rounds of troubleshooting, we discovered that there was accidentally designed two BsaI cutting sites in Pseudomonas fluorescens MT sequence which caused failure of assembly, therefore this part cannot be used in JUMP assembly. Due to Due to failed assembly, we decided to drop Pseudomonas fluorescens MT.
Result and Discussion
Due to failed assembly, Pseudomonas fluorescens MT had sadly been dropped from protein improvement part.
Docking simulation
Non-designed Pseudomonas fluorescens 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 6 Ag+ docked with Gibbs free energy per ion binding of -0.407 kcal/mol. This data was compared with Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Danio rerio and Saccharomyces cerevisiae (Table 1) The data indicates that Pseudomonas fluorescens, which has the fewest number of cysteines, binds the greatest number of ions and having the lowest total Gibbs free energy and Gibbs free energy of binding per ion.This suggests that Pseudomonas fluorescens has a high binding efficiency and affinity for Ag+ ions.
- Figure 2. 3D structure of wilt-type Pseudomonas fluorescens 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]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 627
References
Habjanič, J., Chesnov, S., Zerbe, O. and Freisinger, E., (2019) Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins. Metallomics, 12(1), 23-33.
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.
Olafson, R. W., McCubbin, W. D. and Kay, C. M., (1988) 'Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium', Biochemical journal, 251(3), 691-699.
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 |