ENGINEERING SUCCESS
Research
To create a project for iGEM, our team decided that it is best to approach current issues and tackle them through biological methods. Hence, our team started to look for different global issues. We explored many different issues, mainly focusing on environmental issues.
Imagine
We have noticed that rivers are polluted and fish are dead. As we are interested in the reason behind these problems, our team decided to set up our project revolving around the solution to this problem. After discovering that contamination by palladium (II) ions can be the cause, we read journals to find out how this problem could be resolved. Then, we noticed that current solutions mainly focus on palladium extraction but include no follow-up steps. Thus, to reduce palladium (II) ions to Pd nanoparticles, out team designed palladium-specific reducing peptides.
Design
We decided to incorporate double tryptophan in our peptide sequences. As previous literature has suggested, double tryptophan-based peptides can reduce gold and silver effectively1. As double tryptophan-based peptides for palladium (II) ion reduction have not been investigated, we decided in incorporate it into our design and then evaluate its binding and reducing properties through modelling and wet lab.
Moreover, it has been suggested that a double amino acid substitution of Pd4 library peptide, i.e. H6H11 to A6C11 and C6A11, can enhance its binding ability2. These two amino acid substitutions have comparable binding properties. In the end, we decided to use A6C11 as the anchoring site of our peptide designs.
Build
For preliminary modelling, we used a Bond Fluctuation model (BFM) to simulate the peptide structure and the interaction matrix from literature to simulate the interaction of peptide and the palladium. A python code was written to generate the structure of the peptide.
Test
We tested our python code. While the peptide structure can be generated, the interaction matrix part of the code is not in the stage of testing.
Learn
After the preliminary modelling, we learnt that the Bond fluctuation model has its shortcomings. For example, the Covalent Pd-S (Cysteine) bond is not defined in the interaction matrix. The energetics involving the thiol side chain of the cysteine residue is not included in the bond fluctuation model. Therefore, we have decided to use a more accurate model to simulate the interaction.
Improve
The more accurate method we chose was building a molecular dynamics model of our peptide and the Pd(II) ion, GROMACS (GROningen MAchine for Chemical Simulations) was used as the software package for the simulation because it was the most user-friendly software we could find. GROMACS is a molecular dynamics simulation software used to simulate proteins, lipids and nucleic acids. However, choosing the software was not enough, force fields were also required to be chosen to continue with the simulation. We read journals on running molecular dynamics simulations on metal binding peptides, in which most of them used CHARMM27 or GROMOS 53a5 force field. We decided to use CHARMM27 as the force field because there is more documentation on the parameterization of the force field.
In the end, we succeeded in using GROMACS 2020.3 to test the peptide sequences designed.
Future Direction
All molecular dynamics models built were based on peptide structures with a protonation state in a neutral pH environment. It is commonly known that the pH of the simulation cannot be variated in GROMACS 2020.3. Peptide structures with a protonation state in an acidic or basic environment will be prepared to further analyse the Pd (II) reducing strength of the peptides. More molecular dynamics models will be constructed to give our team deeper insights into the binding and reducing ability of the proposed peptide.
Experiment
We could not preform the experiment physically due to the pandemic so these are the procedures we envisage to carry out. First, DNA sequences coding for our proposed peptide are designed and are amplified using PCR. Gel electrophoresis is then performed to verify the size of the DNA fragments. If correct, the DNA is purified from the gel and then digested by restriction enzymes. If not, PCR amplification and gel electrophoresis are repeated until positive results are obtained. The digested DNA fragments are then ligated into plasmids to be transformed into E.coli cells. Cells containing the target plasmids are identified by testing the cells’ antibiotic resistance and performing a blue white screening test. If the cells fail the screening, meaning that the target DNA were not ligated successfully into the plasmids, the ligation and transformation processes are repeated until positive results are obtained. Colony PCR is then conducted to further confirm that the correct clones containing target plasmids are obtained. At last, the target plasmids are cloned successfully in cloning strains.
References
[1] Si, S., & Mandal, T. K. (n.d.). Tryptophan-Based Peptides to Synthesize Gold and Silver Nanoparticles: A Mechanism and Kinetic Study. doi:10.1002/chem.200601492
[2] Coppage, R., Solcik, J. M., Dakhel, H. R., Bedford, N. M., Heinz, H., Naik, R. R., & Knecht, M. R. (n.d.). Exploiting Localized Surface Binding Effects to Enhance the Catalytic Reactivity of Peptide-Capped Nanoparticles.