Team:NIT Warangal/Contribution

iGEM NIT_Warangal

Adding new documentation to an existing Part on that Part's Registry page

In this challenging time for the whole world, where COVID-19 pandemic has affected millions of life and the way of living and working, it is undoubtedly impacted the iGEM project work as most of us did not have the access of the laboratory throughout the iGEM cycle due to COVID restrictions throughout the nation. So, as part of our contribution, we have added new information from the literature and research papers to the main page of an already existing Part. The Part in which we have added the documentation is "His-tagged laccase, mutated to increase the specificity for
SMX" BBa_K2835004 and this Part was originally added by the team iGEM18_Stockholm, a 2018 iGEM team.

The initial work in the dry lab for our project suggested that the enzyme we are targeting is Laccases and the model organism of our interest is Pichia pastoris. So, searching for any part on the registry which combines both of the above project related terms land us to this part, BBa_K2835004 . In this part, we found that the information about the source organism is missing along with the laccases basic introduction, which is crucial to understand this part in a detailed way. Also, looking for the plasmid, we found that the plasmid vector has not been added to the registry page for this part, so, we also incorporated the restriction map image in our documentation. Later advancing in our project stages, we realised that we will not be using this part in our project directly, but since we know the importance of complete information for any parts, and significant overlapping between this part and our project model, we decided to add the literature information to this part page which will help the future iGEM teams to get more detailed information before using this part. Our contribution to the part BBa_K2835004 will help to gain more knowledge about this part. The information we have added is available below or 0n the main page of this part in the registry.

Trametes versicolor

T. versicolor, which was previously known as Trametes Coriolaceae, is among world top 25 medicinal macrofungal communities^[1]. It is a white-rot fungus capable of degrading lignin in wood. Mycelial colonies of Trametes versicolor were of fade white color and form a high density colony with velvety texture, plenty of aerial hyphae and high growth rate when grown on Malt extract agar and potato dextrose agar supplemented with oak sawdust substrate.

Fig 1: Wild type Trametes versicolor

Source: D. Gonzalez Guerrero et al., Micologia aplicada international(2011)

1GYC (Mutated version of a laccase from Trametes versicolor)

A mutated laccase of Trametes versicolor, which is widely used for it's lignin catabolic activity. It also shows efficient oxidoreductase activity. Here we are showing the structure of 1GYC in ribbon form.

Fig 2: Molecular structure of GYC1, a laccase from Trametes versicolor

Source: https://www.rcsb.org/structure/1GYC

Laccases

Laccases, in general, are copper oxidases found mainly in plants, insects and fungi but some bacterias also secretes this enzyme. It uses oxygen in its native state as co-substrate for the catalysis and yields water as the sole by-product after the whole reaction hence it is very popular, widely used for lignin degradation activity even though it has a significant lignin formation activity as well.It oxidizes various phenolic compounds by one electron oxidation method. Fungal laccases are involved in a wide range of natural phenomenon like sporulation, pigment production, fruiting body formation, stress defense, plant pathogenesis, and lignin degradation^[2]. Laccases, a classified benzenediol oxygen reductases , are members of the cupredoxin superfamily and belongs to a subfamily multi-copper oxidases (MCOs). They have four copper atoms in remarkably special oxidation states: one type-1, one type-2, and two type-3s, all forming their catalytic site in the molecule which is shown in figure below:

Fig 3: 1GYC copper binding residues that are matched with a defined pattern, shown as T1, T2 AND 2-T3.

Source: Sirim et al., Database : the journal of biological databases and curation. (2011)

We have also identified the various restriction enzyme sites present in the gene sequence of the given laccase as it was not available in the part plasmid vector. We used the NEB cutter tool to generate this labelled image.

Fig 4: Gene sequence of 1GYC(mutated laccase gene) with identified restriction enzyme sites in it.

Addition of New Parts

We have added some new parts to the registry page which will be available there for the use of future iGEM teams. So, it is indeed categorised as the contribution to the iGEM community where our designed parts are being available for the use of the global community through an open source channel.
BBa_K4077000 was added as a basic part and is a laccase found in a fungus known as Melanocarpus albomyces. This sequence was optimised by GenScript for expression in Pichia pastoris plasmid.

In the vector, for efficient protein folding in the endoplasmic reticulum, BBa_K4077005 was genetically engineered. This is known as an alpha-factor secretion signal that is isolated from Saccharomyces cerevisiae. It was added to the registry in the basic part category.

The last basic part was BBa_K4077003 , the plasmid that we used for our experiments. It is a modified Pichia pastoris expression vector (pPIC9K).

Finally, the composite part we added to the registry was, BBa_K4077008, which is the laccase gene insert, including promoter and terminators. It included the subparts, BBa_K3196036 and BBa_K3196026, from iGem 2019 team HUST China which were the AOX1 promoter and terminator sequences respectively. The AOX1 promoter sequence is isolated from the Pichia pastoris genome and genetically engineered into the pPIC9K expression vector.

A Guide to Idea Generation for Future iGEM Teams

Idea generation and narrowing it down to a project idea is a crucial step for research, especially for a research-based competition, where different teams participate and work on a project to solve a problem.

What if the teams could not narrow down their ideas to a possible project idea that is practical according to the present technology.

If you are also stuck, don't worry iGEM NIT Warangal is here to rescue you.

It takes a lot of brainstorming and time. To help you be efficient in your idea generation process, we have created a small booklet that will help your team narrow down ideas to a possible project idea.

There are three sources of ideas: local problems, existing scientific research papers, and inspiration from past iGEM teams. We have combined all three sources and transformed them into a handbook that would benefit iGEM Teams during their idea generation process.

Handbook

AutoDock protocol

This is another important contribution to the iGEM community where we are providing a manual about how to do AutoDocking in simple steps. While drug design and testing, researchers use AutoDock for various purposes like screening, viewing the cavities, etc. It is a widely used software, which makes learning crucial.

For beginners, we have brought AutoDock protocols. This is a list of steps to be followed while you use AutoDock. It also consists of basic steps like removing water molecules and adding hydrogen, which play a crucial role.

Protocol for exporting protein-Ligand docked complex from AutoDock 4.2

In order to export the protein-ligand docked complex from AutoDock 4.2, follow this procedure:

Open the protein ".pdbqt" file in AutoDock 4.2 by going to File in the Navigation bar, and clicking on Read molecule in the drop down menu. Select the required file.
Once the protein file is opened, go to Analyze --> Macromolecule --> Choose, and select the protein file in the dialog box that appears.
Now go to Analyze --> Dockings --> Open, and open the ".dlg" file (docking log file), which is obtained after successful protein-ligand docking
Once the docking log file is opened go to Analyze --> Conformations --> Play ranked by energy. A panel with play options appears. The first conformation that appears is the one with the best docking score.
Navigate to the required ligand conformation and click on this button: . A panel to change play options appears.
Check the "Build H-bonds" in the panel (optional), and click on "Write Complex". The complex can be saved in a ".pdbqt" format and used for further analysis via other tools.

We have also summarised the AutoDock protocol for preparing macromolecules, ligands and stepwise procedure for docking and autogrid process. Check the file attached below for getting the protocols.

AutoDock Protocol

Protocol to analyse ligand interactions in Discovery Studio

Open the complex file of the desired molecule in Discovery Studio under Receptor-Ligand Interactions
Remove unwanted parts(in this case , chain B and Heatatm , etc.) to keep only binding site and essential amino acids
Now, click on Ligand Interactions to visualize the interactions between ligand and molecule in a 3D format.
To observe interactions in a 2D format, click on show 2D diagram.

Protocol for Energy Minimization of an enzyme in Chimera 1.15

Open the protein file in UCSF Chimera which is to be energy minimized.
Go to Tools in the Navigation bar and select Structure editing from the dropdown menu. Select Minimize Structure and a dialog box appears.
We used the number of steepest descent steps as 100 and their size as 0.02 A^o. The number of conjugate gradient steps was 100 and their size was 0.02 A^o. The update interval we used was 10 with no fixed atoms. These are the recommended settings for energy minimization.
Click on minimize, and another dialog box appears. Check the "consider each model in isolation from all others" box. It is highly recommended to also consider H-bonds under Method, although it makes the process slower. Use protonation states for histidine. Keep the naming as residue-name-based unless there is need to specify the names individually.
Click OK and wait for hydrogens to be added. A third dialog box appears. Use AMBER ff14SB for standard residues and AM1-BCC for other residues. Click OK and the energy minimization process starts.
Once completed, the amount of energy minimized can be viewed in the Reply Log which can be accessed through Favorites in the Navigation bar.

End is the beginning: iGEMers of 2021 and their diary pages

iGEM is a worldwide growing community of synthetic biology enthusiasts and it's enormous popularity among students fascinates those young minds to come up with new ideas that hold the potential to get scaled up for human use and create a better future altogether. Every year, more and more new teams join this community and participate in this globally renowned competition. But this journey is full of roller coasters and things become tough when someone participates for the first time. Though iGEM itself is an open community full of resources and also provides mentors later on, but in the initial phases and to get the real idea of the whole iGEM journey as in person, we come up with our contribution to the iGEM community where we will build an iGEM diary through collaborative effort of iGEMers around the globe.

What if the teams could not narrow down their ideas to a possible project idea that is practical according to the present technology.

If you are also stuck, don't worry iGEM NIT Warangal is here to rescue you.

Since the wiki will again be activated after jamboree, we have planned to interview the new iGEM teams of this year and will summarize their whole iGEM experience, how have they balanced their academics along with iGEM cycle, what are the challenges they faced as a new iGEM team and how have they overcome these challenges. It will help the future beginners to plan their journey beforehand so that they sail smoothly afterwards. This manual will be active and accessible to everyone even after the wiki freeze and it will get in the final version after the end of this competition. It is so because the iGEMers all around the world are tightly packed with lab works and other wiki related documentation and find it difficult to participate in this initiative. The pdf version will be passed to the next wave of aspiring iGEMers as an open source material. We are also considering distributing it to other iGEM teams of this year to translate it to their native languages so that it reaches a broader section of the science population and beginners in any part of the world will find it helpful and relatable.

This will be a great contribution towards building a realistic iGEM picture in young minds where they can learn and get motivation from others' experience of the iGEM journey and the path they traced to complete the whole iGEM cycle beautifully. We will also pass this concept to our next year iGEM NIT_Warangal team who will follow this trend to build a huge resource for the global community in the name of iGEMers diary pages.

From Animal Lovers to Animals

There is a lack of food source for stray and domestic animals due to the breakdown of the COVID-19 pandemic. Animal lovers are worried about the devastating situation of animals.

To help these animal lovers feed the animals, especially the ruminants and to rescue the animals from such a devastating situation we have come up with a low cost, effective and protein rich cottonseed meal.

Regardless of their project and their track selection, other iGEM teams have shown their interest in our project and in saving the animals. Animals need a reliable food source! They need readily available and inexpensive sources of nutrition, they need the cottonseed meal!

This iGEM wiki itself as a platform to build a food product later on or as a base to think for affordable nutritious food product is our gift to the iGEM community and for the animals, the reason why we tried to solve the nutritional problem. We spent hours and sleepless nights working on it and now it's time to donate this to the future iGEMers for a better tomorrow!

Stay safe and take care of animals!

Yours,
Team iGEM NIT Warangal

References:

1. Gonzalez Guerrero, D., Esparza Martinez, V. and Torre Almaraz, R. from the CULTIVATION OF TRAMETES VERSICOLOR IN MEXICO. Applied Mycology International. 2011; 23 (2): 55-58.
Source: https://www.redalyc.org/articulo.oa?id=68519085003
2. Arregui, L., Ayala, M., Gomez-Gil, X. et al. Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact 18, 200 (2019).
DOI: https://doi.org/10.1186/s12934-019-1248-0
3. With no source of food, animals suffer in silence by Rajulapudi Srinivas, from The Hindu