iGEM 2023 — Team plknplc-hk

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{% block title %}Notebook{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
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Optimization of selected proteins

#### Design:

In addition to finding the enzymes with the most endogenous decolourizing ability for MB, we also wanted to optimise the activities of the enzymes which showed the highest percentage of decolourization.

The structure for BBa\_K4936001, a Trametes versicolor lignin peroxidase isozyme, was predicted using Alphafold. Chiron, a server provided by Dokholyan Lab at the Pennsylvania State University, was used for further energy minimization of the structure. Crystal structures for our other proteins were retrieved from the PDB database.

We then applied the Alphafill algorithm to reconstitute the protein cofactors with homology based prediction. We then cleaned the structures with pdb-tools, a pypi package publicly available on Github by bonvin lab [[1]](https://doi.org/10.12688/f1000research.17456.1)

#### Build:

Due to time constraints, we were unable to construct and introduce our protein mutants to a biological chassis to analyze their activities in vivo. The in silico tests we were able to perform are detailed below.

#### Test:

Protein docking for all protein structures was performed by SwissDock [[2]](https://doi.org/10.1093/nar/gkr366) with accurate presets and 3Å sidechain binding pocket flexibility. Further analysis was performed by UCSF Chimera [[3]](https://doi.org/10.1002/jcc.20084) and visualised in PyMOL. Preliminary protein-ligand interaction analysis was performed in Discovery Studio and ligand coordinates were then extracted from the protein-ligand complex for Molecular Dynamics Simulation in GROMACS. Note that hydrogen atoms were added in OpenBaBel [[4]](https://doi.org/10.1186/1758-2946-3-33), prior to parameterization and topology generation in CGenFF [[5]](https://doi.org/10.1002/jcc.21367).

#### Learn:

We have gained a thorough understanding of the optimal methods, parameters, and tools for structure optimization and analysis. To expand on our newfound knowledge and generated data, we plan to do further analysis on our structures to approach the issue of methylene blue removal from different angles. For example, we originally planned to further identify the best proteins of choice with umbrella sampling to test the activity of less likely conformations, and visualising and characterizing their ability on the molecular level with the QMMM/MD functions of the CP2K GROMACS software package.

Our work could also be expanded on by trying to engineer new proteins based on our optimisation data and structures. One possible path is by using flex\_DDG [[6]](https://doi.org/10.1021/acs.jpcb.7b11367) to generate a wide sample of mutated structures and model their changes in binding free energies.

We also plan to validate the efficiency of different media in supporting the growth of S. elongatus under conditions in which our proteins of interest or heme synthesis genes are overexpressed. A suitable candidate is the BG-11 medium modified by HK\_SSC 2021 [[7]](https://2021.igem.org/Team%3AHK_SSC/Engineering) with a higher manganese concentration.

##### Citations

[1] Rodrigues JPGLM, Teixeira JMC, Trellet M and Bonvin AMJJ. pdb-tools: a swiss army knife for molecular structures. F1000Research 2018, 7:1961. https://doi.org/10.12688/f1000research.17456.1

[2] Grosdidier, A., Zoete, V., & Michielin, O. (2011). SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic acids research, 39(suppl\_2), W270-W277. https://doi.org/10.1093/nar/gkr366.

[3] Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF Chimera—a visualization system for exploratory research and analysis. Journal of computational chemistry, 25(13), 1605-1612. https://doi.org/10.1002/jcc.20084.

[4] O'Boyle, N.M., Banck, M., James, C.A. et al. Open Babel: An open chemical toolbox. J Cheminform 3, 33 (2011). https://doi.org/10.1186/1758-2946-3-33

[5] Vanommeslaeghe, K., Hatcher, E., Acharya, C., Kundu, S., Zhong, S., Shim, J., ... & Mackerell Jr, A. D. (2010). CHARMM general force field: A force field for drug‐like molecules compatible with the CHARMM all‐atom additive biological force fields. Journal of computational chemistry, 31(4), 671-690. https://doi.org/10.1002/jcc.21367.

[6] Barlow, K. A., Ó Conchúir, S., Thompson, S., Suresh, P., Lucas, J. E., Heinonen, M., & Kortemme, T. (2018). Flex ddG: Rosetta ensemble-based estimation of changes in protein–protein binding affinity upon mutation. The Journal of Physical Chemistry B, 122(21), 5389-5399. https://doi.org/10.1021/acs.jpcb.7b11367.

[7] https://2021.igem.org/Team:HK\_SSC/Engineering

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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## Results

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This year, our team has focused on complementary in silico and in vitro analysis of our selected proteins of interest for Methylene Blue (MB) degradation.

Previous studies on elucidating the molecular mechanism of biodegradation by ligninolytic enzymes have suggested the diversity of active binding sites for different common commercial dyes such as Congo Red and Methyl Orange [[4]](https://www.researchgate.net/publication/362278809_UNDERSTANDING_ENZYME-LINKED_BIODEGRADATION_BY_MOLECULAR_DOCKING_OF_SCHIZOPHYLLUM_COMMUNE%27S_LACCASE_LIGNIN_PEROXIDASE_AND_MANGANESE_PEROXIDASE_WITH_COMMERCIAL_DYES). In order to gain a deeper understanding of the molecular mechanisms of MB biodegradation by our proteins of interest, we performed in silico analysis with GROMACS molecular dynamics simulation.

First and foremost, we did protein structure preparation, which is the most important aspect of in silico analysis. High-resolution X-ray crystallography-resolved structures were selected from the RCSB Protein Database. For instance, the 0.93 Å structure of Phanerodontia chrysosporium Magnesium Peroxidase (PDB ID:3M5Q) was used in one of our analyses to ensure the validity of our GROMACS molecular dynamics simulation.

Apart from protein structures obtained from the RCSB Protein Database, the protein structure of a newly characterized Trametes versicolor lignin peroxidase isozyme (LPG3) was prepared with ColabFold, a multiple sequence alignment (MSA)-mediated AlphaFold protein structure prediction tool [[3]](https://doi.org/10.1038/s41592-022-01488-1). Cofactors were reconstituted into the protein structure by AlphaFill, a homology-based ligand transplantation algorithm [[2]](https://doi.org/10.1038/s41592-022-01685-y).

MB-docked structures were prepared with SwissDock and analysed with UCSF Chimera prior to GROMACS Molecular Dynamics Simulation, for which the simulation results are shown below.

Our in silico analyses have demonstrated the MB degradation potential of all proteins of interest as suggested by our data on protein-ligand binding and interaction modes, supporting in vivo ability to form complexes with MB. This has prompted our plans to perform further analyses, such as with Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) to further decouple the energetics within the system, or co-incubation and validation with cryo-EM in wet lab.

Overall, the selection of all 5 proteins as potential candidates to proceed to in vitro analysis was based on literature review and our in-silico analysis workflow.

#### Wet Lab

The idea of building a Synechococcus elongatus shuttle vector first began with iGEM Team Marburg 2019’s shuttle vector BBa\_K3228089. This plasmid backbone was further modified by Team HK\_SSC 2021 to create BBa\_K3776009. Our team has now designed our plasmid based on these two previous contributions in view of their modularity and compatibility with our selected chassis Synechococcus elongatus UTEX 2973.

![](https://static.igem.wiki/teams/4936/wiki/results/liph8-plasmid-2.jpeg)

Figure 1: lph8 plasmid

![](https://static.igem.wiki/teams/4936/wiki/results/lac2-plasmid-2.jpeg)

Figure 2: lac2 plasmid

![](https://static.igem.wiki/teams/4936/wiki/results/lpg-3-plasmid-2.jpeg)

Figure 3: lpg3 plasmid

![](https://static.igem.wiki/teams/4936/wiki/results/mnp1-plasmid-2.jpeg)

Figure 4: mnp1 plasmid

![](https://static.igem.wiki/teams/4936/wiki/results/vpl2-plasmid-2.jpeg)

Figure 5: vpl2 plasmid

![](https://static.igem.wiki/teams/4936/wiki/results/gel-photo-labeled-1.png)

Figure 6: DNA gel electrophoresis showing the PCR amplification results. All DNA bands correspond to the genes of interest.

lac2: 1KYA Trametes versicolo Laccase

mnp1: 3M5Q Phanerodontia chrysosporium Magnesium Peroxidase

liph8: 1B80 Phanerodotia chrysosporium Lignin Peroxidase H8

lpg3: TvLiP Trametes versicolo Lignin Peroxidase

vpl2: 2BOQ Pleurotus eryngii Versatile Peroxidase

After molecular cloning, further analysis on protein expression will be performed based on protocols contributed by iGEM Team HK\_SSC 2021 and HK\_SSC 2022.

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#### Dry Lab

The interactions between Methylene Blue and Proteins of interest are analyzed by Discovery Studio [[1]](https://discover.3ds.com/discovery-studio-visualizer-download), and visualized by PyMOL (Schrodinger LLC, 2020). Note that MB has different binding sites among all proteins of interest.

The above analysis serves as preliminary models to identify and visualize interactions between Methylene Blue and Proteins of interest.

#### 2BOQ: *Pleurotus eryngii* Versatile Peroxidase (vpl2) ![](https://static.igem.wiki/teams/4936/wiki/results/2boq-2d.jpeg) In brief, Hydrogen Bonds are formed between MB and R119, Q255, P285, and P286. π-alky interactions are formed between MB and V118 and A259. ![](https://static.igem.wiki/teams/4936/wiki/results/2boq-3d.png) 3M5Q: *Phanerodontia chrysosporium* Magnesium Peroxidase (mnp1) ![](https://static.igem.wiki/teams/4936/wiki/results/3m5q-2d.jpeg) van der Waals interactions contributed by T193,T196, T199, Q262, A299, T300, G301 and Q302 are present in the binding pocket. Hydrogen bonds are formed between MB and Q200, N260 and A304. Alkyl interactions are formed between MB and P303 and M305. π-anion interactions are present between MB and D198. ![](https://static.igem.wiki/teams/4936/wiki/results/3m5q-3d.png) 1KYA: Trametes versicolor Laccase (lac2) ![](https://static.igem.wiki/teams/4936/wiki/results/1kya-2d.jpeg) van der Waals interactions contributed by L35, P123, and N 172 are present in the binding pocket. Hydrogen bonds are formed between MB and D142 and V145. π-alky interactions are formed between MB and P32. ![](https://static.igem.wiki/teams/4936/wiki/results/1kya-3d.png) 1B80: *Phanerodotia chrysosporium* Lignin Peroxidase H8 (liph8) ![](https://static.igem.wiki/teams/4936/wiki/results/1b80-2d.jpeg) van der Waals interaction contributed by I199 is present in the binding pocket. Hydrogen bonds are formed between MB and I295 and F301. π-alky interactions are formed between MB and I295 and L299. π-Sigma interactions are present between MB and I295. ![](https://static.igem.wiki/teams/4936/wiki/results/1b80-3d.png) TvLiP: *Trametes versicolor* Lignin Peroxidase (lpg3) ![](https://static.igem.wiki/teams/4936/wiki/results/tvlip-2d.png) Hydrogen bonds are formed between MB and P110, H109 and E173. π-alky interactions are formed between MB and V213 and P215. ![](https://static.igem.wiki/teams/4936/wiki/results/tvlip-3d.png) [1] BIOVIA, Dassault Systèmes, BIOVIA Discovery Studio Visualizer, Version 21.1.0.20298, San Diego: Dassault Systèmes, 2020. [2] The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC. ---

#### The SwissDock binding mode cluster with lowest FullFitness Energetics is shown below 2BOQ: Pleurotus eryngii Versatile Peroxidase (vpl2) ![](https://static.igem.wiki/teams/4936/wiki/results/2boq-energetics-2.png) 3M5Q: Phanerodontia chrysosporium Manganese Peroxidase (mnp1) ![](https://static.igem.wiki/teams/4936/wiki/results/3m5q-energetics-2.png) 1KYA: Trametes versicolor Laccase (lac2) ![](https://static.igem.wiki/teams/4936/wiki/results/1kya-energetics-2.png) 1B80: Phanerodontia chrysosporium Lignin Peroxidase H8 (liph8) ![](https://static.igem.wiki/teams/4936/wiki/results/1b80-energetics-2.png) TvLiP: Trametes versicolor Lignin Peroxidase (lpg3) ![](https://static.igem.wiki/teams/4936/wiki/results/tvlip-energetics-2.png) Note that the Ligand Binding Mode with the lowest FullFitness Energetics in the cluster is utilized for subsequent GROMACS Molecular Dyamics Simulation Analysis. In brief, FullFitness energetics takes into account the internal energy of the ligand and the enzyme, the interaction between the ligand and enzyme complex and the solvation energy by electrostatic ,and non-polar contributions proportional to the solvent accessible surface area (SASA) [[1]](https://doi.org/10.1002/prot.21367). ![](https://static.igem.wiki/teams/4936/wiki/results/fullfitness-energetics-equation.png)

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#### Reference: [1] Grosdidier, A., Zoete, V., & Michielin, O. (2007). EADock: Docking of small molecules into protein active sites with a multiobjective evolutionary optimization. Proteins, 67(4), 1010–1025. https://doi.org/10.1002/prot.21367 [2] Hekkelman, M. L., De Vries, I., Joosten, R. P., & Perrakis, A. (2022). AlphaFill: enriching AlphaFold models with ligands and cofactors. Nature Methods, 20(2), 205–213. https://doi.org/10.1038/s41592-022-01685-y [3] Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Овчинников, С. Г., & 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 [4] Sorhie, V., Chang, C., Bharali, P., & Bhaskar, M. (2022). UNDERSTANDING ENZYME-LINKED BIODEGRADATION BY MOLECULAR DOCKING OF SCHIZOPHYLLUM COMMUNE’S LACCASE, LIGNIN. . . ResearchGate. https://doi.org/10.14704/nq.2022.20.6.NQ22791

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