Part:BBa_K4763007
AtPCS (phytochelatin synthase) coding sequence
Phytochelatin synthase (AtPCS) coding sequence from Arabidopsis thaliana. This enzyme catalyzes the final step in the biosynthesis of phytochelatins (PCs) using as a co-substrate the toxic heavy metal cadmium.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 649
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 649
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 649
- 1000COMPATIBLE WITH RFC[1000]
Contents
Usage and Biology
The enzyme chosen for the generation of the biopart was phytochelatin synthase or PCS (EC 2.3.2.15), which catalyzes the synthesis of glutathione (GSH) polymers called phytochelatins (PCs) in presence of toxic heavy metals (Figure 1). These are produced as a mechanism of resistance and accumulation in algae, yeast, plants, and worms in response to cadmium stress, mainly. Phytochelatins bind to metals intracellularly and render them inactive. This is because the substrate of PCS is GSH (as aforementioned) and other metal-bis-glutathione complexes. Additionally, the enzyme activity requires the presence of specific heavy metals (depending on the species under study), with cadmium being the main inducer in plants and other organisms. The complexes formed by the metal and PCs can be subsequently compartmentalized into vacuoles, chloroplasts, and mitochondria (García-García et al., 2014; 2020).
The PCS enzyme from the model organism Arabidopsis thaliana (AtPCS1) was selected for the creation of this biopart due to its more extensive characterization in the synthesis of phytochelatins.
Characterization
Molecular dynamics
A predicted structure was obtained through ColabFold (Mirdita et al., 2022) using existing crystallographic structures for each constituent protein (Figure 2). To lend more confidence to the predicted structure, molecular dynamics (MD) simulations of the obtained model were performed. This helped assess if the predicted structure was maintained after the protein was exposed to biophysical potentials.
MD simulations were performed to assess if the predicted structure obtained of the system was conserved after including biophysical potentials. A preliminary simulation was run with GROMACS using model 5 from ColabFold. The topology was generated with the force field AMBER99SB-ILDN and the water model TIP3P (Figure 3).
Cloning AtPCS insert into pET28(+) vector
The gene sequence for AtPCS was transformed into the pET28b(+) vector. This was done using T4 DNA Ligase (New England Biolabs). Table 1 shows the components used for the ligation reaction at a 1:7 molar ratio:
| Reagent | Quantity |
|---|---|
| Insert | 192 ng (4.9 µL) |
| T4 DNA ligase buffer | 2 µL |
| Vector | 100 ng (2.6 µL) |
| T4 DNA ligase | 1 µL |
| Nuclease-free water | 9.5 µL |
The ligation was then transformed into E. coli TOP10 by adding 10 μL of the ligation reaction to 50 μL of competent cells following the heat shock method. After incubation colonies were observed indicating successful transformation (Figure 4).
Screening of transformed cells by colony PCR
To verify the presence of AtPCS gene in the pET28b(+) plasmids, a colony PCR was performed with five transformed colonies, where a band was observed near the 1500 bp mark, which matches the length of the gene (1461 bp) (Figure 5).
Restriction enzyme digestion test
To identify, through an additional methodology, the clones that were correctly transformed with the gene, restriction assays were performed on plasmids obtained from colonies 1-3 using the NdeI and EcoRI-HF enzymes. Table 2 displays the protocol followed for a 50 µL reaction.
| Reagent | Quantity |
|---|---|
| Nuclease-free water | add to 50 µL |
| rCutSmart Buffer | 5 µL |
| AtPCS_pET28b(+) candidates | 400 ng (10 µL) |
| NdeI restriction enzyme | 1 µL |
| EcoRI restriction enzyme | 1 µL |
Figure 6 shows the agarose gel with the results of the enzymatic digestions, where a band corresponding to the length of the AtPCS gene was identified in sample D1 from colony 1.
Protein overexpression trials in E. coli BL21
Overexpression trials were performed by induction with 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cell lysis was performed through sonication and intracellular protein presence and concentration was measured through an SDS-PAGE gel, as shown in Figure 7. The gel showed a very faint band present at the appropriate weight for the AtPCS gene.
References
[1]. García-García, J. D., Sánchez-Thomas, R., Saavedra, E., Fernández-Velasco, D. A., Romero-Romero, S., Casanova-Figueroa, K. I., … Moreno-Sánchez, R. (2020). Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase. Algal Research, 101890. doi:10.1016/j.algal.2020.101890
[2]. García-García, J. D., Girard, L., Hernández, G., Saavedra, E., Pardo, J. P., Rodríguez-Zavala, J. S., Encalada, R., Reyes-Prieto, A., Mendoza-Cózatl, D. G., & Moreno-Sánchez, R. (2014). Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis. Metallomics : integrated biometal science, 6(3), 604–616. https://doi.org/10.1039/c3mt00313b
[3]. Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nature methods, 19(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1
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