iGEM 2021 — Team NIT_Warangal

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# Team:NIT Warangal/Notebook

iGEM NIT\_Warangal

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* ![](https://static.igem.org/mediawiki/2021/7/73/T--NIT_Warangal--icons--NAVBARpresentation.png)PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3ANIT_Warangal/Description)
  [ENGINEERING](https://2021.igem.org/Team%3ANIT_Warangal/Engineering)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANIT_Warangal/Implementation)
  [PARTS](https://2021.igem.org/Team%3ANIT_Warangal/Parts)
  [NOTEBOOK](https://2021.igem.org/Team%3ANIT_Warangal/Notebook)
* ![](https://static.igem.org/mediawiki/2021/f/f1/T--NIT_Warangal--icons--NAVBARlab.png)LAB WORK

  [LAB](https://2021.igem.org/Team%3ANIT_Warangal/Lab)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANIT_Warangal/Experiments)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept)
  [RESULTS](https://2021.igem.org/Team%3ANIT_Warangal/Results)
  [SAFETY](https://2021.igem.org/Team%3ANIT_Warangal/Safety)
* ![](https://static.igem.org/mediawiki/2021/e/e9/T--NIT_Warangal--icons--NAVBARsocial.png)SOCIAL

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANIT_Warangal/Human_Practices)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANIT_Warangal/Collaborations)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANIT_Warangal/Contribution)
  [COMMUNICATION](https://2021.igem.org/Team%3ANIT_Warangal/Communication)
  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3ANIT_Warangal/Entrepreneurship)
* ![](https://static.igem.org/mediawiki/2021/b/b8/T--NIT_Warangal--icons--NAVBARtest.png)JUDGING

  [MEDALS](https://2021.igem.org/Team%3ANIT_Warangal/Medals)
  [JUDGING FORMS](https://igem.org/2021_Judging_Form?id=4077)

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# January

**20th January -** Formation of a core team.

 **31st January -** Meet with BITS Goa - they gave us a small introduction to the competition and showed us their wiki.

# February

**6th February -** Meet with IIT Roorkee - Discussions with them regarding funding and their team plan for the previous year.

 **22nd February -** All team members are finalized.

 **23rd February -** We contact our advisors to join the team.

 **24th February -** Entire team meet where all members interact with PI and the advisors - advisors give suggestions on choosing an idea.

 **25th February -** We started brainstorming ideas.

# March

**1st March -** All team members come with their ideas and give a small presentation on the topic and potential solutions.

 **6th March -** Entire team meet to explore wikis of past teams.

 **10th March -** Team narrows down to five topics and three members each work on a particular topic.

 **11th March -** Team contacts HoD of Biotech Dept of NITW to intimate him regarding the project.

 **15th March -** Team narrows down to 2 ideas - soil nutrient biosensors and cottonseed degossypolization.

 **17th March -** Meet with iGEM India Ambassador, Shruti Sridhar to gain proper insight about iGEM and what to expect throughout the competition.

 **21st March -** Finalising the Team structure.

 **25th March -** Meet with Dr.Mahesh from the ECE Dept of NITW, who provided inputs regarding the feasibility of biosensors for soil nutrients.

# April

**3rd April -** Meet with IISER Berhampur iGEM team of 2020 to discuss how to finalize an idea and talk about their experience of iGEM.

 **7th April -** Decided upon the problem that we aim to solve - gossypol.

 **10th April -** Funding Campaign starts.

 **17th April -** FINAL PROJECT PLAN CONFIRMED.

 **18th April -** iGEM committee set up in college.

 **19th April**  to **30th April -** All members participate in a full-fledged public funding campaign.

# May

**3rd May -** Started going through literature on gossypol toxicity.

 **8th May -**Finalized the project brochure for public distribution.

 **12th May -** Finalized the project brochure for companies.

 **17th May -** Had our first meeting with our mentor Avadhoot Jadhav.

 **18th May -** Secured the funds required for registration to the competition.

 **20th May -** Presented the final outline of the project to the advisors.

 **21st May -** Made a lab notebook for project documentation.

 **24th May -** Finalized template for team introduction on social media.

 **25th May -** Team leader attended a pan India iGEM Meet.

 **26th May -** Team completes literature review.

 **28th May -** Registered team for the iGEM competition.

 **30th May -** Found the first batch of potential enzymes.

# June

**5th June -** Decided upon the basic plan of action of the project.

 **6th June -** Used Pharmmaper and started exploring docking software.

 **8th June -** Discussed Pharmmaper results.

 **9th June -** Attended the Safety Workshop conducted by iGEM Asia Ambassadors.

 **10th June -** Attended a small workshop on PyMOL.

 **12st June -** Dry lab team meets to discuss regarding CB-Dock server.

 **14th June -** Decided to reject pharmmapper results and look for other software.

 **17th June -** Entire team meet with Advisory Committee to discuss about CB-Dock.

 **18th June -** Team starts screening using CB-Dock.

 **19th June -** Have a meet with Avadhoot(iGEM mentor) to discuss about our project.

 **20th June -** Team fills preliminary safety form.

 **24th June -** Autodock workshop conducted by Santanu (our Advisor) sir for all dry lab members.

 **25th June -** Had an in-depth discussion regarding protocol for autodock.

 **26th June** to **30th June -** All members familiarise themselves with Autodock Software.

 **29th June -** Stitch software is suggested by our secondary PI as another potential screening tool.

# July

**3rd July -** Meet with Dr.Keerti Rathore.

 **5th July -** We start preparing the project promotion video.

 **7th July -** STITCH is rejected.

 **12th July -** 10 preliminary enzymes are finalized.

 **15th July -** 2nd preliminary enzyme list is discussed.

 **16th July -** 500 runs Autodock on 5 enzymes.

 **21st July -** Approached by NextGen Scientists' Foundation for team interview.

 **23rd July**  to **25th July -** All India iGEM Meet.

 **25th July -** Presented at the meet.

 **27th July -** Meet with the Advisory Committee to decide upon wet lab experiments.

 **31st July -** Meet with IISER\_TVM to discuss docking and protein modeling software.

# August

**2nd August -** Team starts industry analysis of cotton sector in India.

 **3rd August -** The team starts discussing Alpha-Fold 2 for protein modeling.

 **7th August -** Team decides workflow for mutagenesis work.

 **8th August -** Team learns how to use Discovery Studio.

 **9th August -** Rolled out a survey for food and nutrition.

 **10th August -** Team learns how to use UCSF Chimera.

 **12th August -** Team learns about Ramachandran Plot Analysis.

 **13th August -** Team starts Giant Jamboree funding.

 **14th August -** Some of our team members have visited local cotton farms and interacted with cotton farmers.

 **15th August**  to **23th August -** Team does mutagenesis work.

 **17th August -** Drafted the basic outline for a symposium on Food Biotechnology.

 **19th August -** Team logo is finalised.

 **24th August -** Giant Jamboree Funding conducted full team.

 **26th August -** Presented mutagenesis results to the Advisory Committee.

 **27th August -** Final enzyme is decided

# September

**2nd September -**Giant Jamboree Funding secured.

 **5th September -** AlphaFold not possible due to low RAM and experienced a crash in results.

 **8th September -** Team tries once more on Alpha Fold but still the same issue.

 **9th September -** Registered for Giant Jamboree.

 **10th September -**Meet with Avadhoot regarding updates.

 **11th September -** Team leader attends Ambassador Cohort to talk about Giant Jamboree.

 **12th September -**Wet lab experimentation and workflow finalized.

 **13th September -** AlphaFold rejected since results are not possible.

 **14th September -** Received codon-optimized script from GenScript.

 **15th September -** iGEM vector journal article finalized.

 **16th September -** Some of our team members have visited a nearby cottonseed oil processing industry

 **17th September -** Meet with team iGEM Alma.

 **19th September -** Wiki design starts.

 **20th September -** SWISS-MODEL and RaptorX protein modelling.

 **21st September -** Finalized Team Roster.

 **23th September -** Food and Nutrition Track finalized on iGEM website.

**24th September -** Presentation Video Script and Preparation is started.

**28th September -** Submitted abstract and title of the project.

 **30th September -** Submitted final Safety Form.

# October

**1st October -** Preliminary Judging form submission.

 **3rd October -** Wet lab work: cloning and expressions.

 **10th October -** Meet with Surbhi Guha for Cost Analysis.

 **11th October -** Finalized "Cos DeGos" name for the project.

 **12th October -** Started the groundwork for the presentation video.

 **14th October -** Meet with MIT\_Mahe for feedback on their website for generating CAD model.

 **16th October -** Put out a feedback form for our project.

 **17th October -** Meet with IISER Mohali for business analysis.

 **17th October -** Meet with IISc Bangalore.

# Sponsors

[![etl_logo](https://static.igem.org/mediawiki/2021/5/5a/T--NIT_Warangal--common--sponsorsLogos--etl_logo.png)](https://emmessartech.org/)

[![reticulum_logo](https://static.igem.org/mediawiki/2021/4/4d/T--NIT_Warangal--common--sponsorsLogos--reticulum_logo.svg)](https://reticulum-community.org/)

[![benchling_logo](https://static.igem.org/mediawiki/2021/3/35/T--NIT_Warangal--common--sponsorsLogos--benchling_sponsor.png)](https://www.benchling.com/)

[![biolabs_logo](https://static.igem.org/mediawiki/2021/1/14/T--NIT_Warangal--common--sponsorsLogos--footer_biolabs_sponsor.png)](https://international.neb.com/)

[![addGene_logo](https://static.igem.org/mediawiki/2021/7/75/T--NIT_Warangal--common--sponsorsLogos--footer_addGene_sponsor.svg)](https://www.addgene.org/)

[![idt_logo](https://static.igem.org/mediawiki/2021/8/89/T--NIT_Warangal--common--sponsorsLogos--footer_idt_sponsor.svg)](https://www.idtdna.com/)

[![snapgene_logo](https://static.igem.org/mediawiki/2021/a/a6/T--NIT_Warangal--common--sponsorsLogos--snapgene_logo.png)](https://www.snapgene.com/)

[![twist_logo](https://static.igem.org/mediawiki/2021/4/41/T--NIT_Warangal--common--sponsorsLogos--twist_logo.png)](https://www.twistbioscience.com/)

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---

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iGEM NIT Warangal

 National Institute of Technology, Warangal

 Warangal, Telangana, INDIA - 506004

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# Team:NIT Warangal/Engineering

iGEM NIT\_Warangal

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* ![](https://static.igem.org/mediawiki/2021/7/73/T--NIT_Warangal--icons--NAVBARpresentation.png)PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3ANIT_Warangal/Description)
  [ENGINEERING](https://2021.igem.org/Team%3ANIT_Warangal/Engineering)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANIT_Warangal/Implementation)
  [PARTS](https://2021.igem.org/Team%3ANIT_Warangal/Parts)
  [NOTEBOOK](https://2021.igem.org/Team%3ANIT_Warangal/Notebook)
* ![](https://static.igem.org/mediawiki/2021/f/f1/T--NIT_Warangal--icons--NAVBARlab.png)LAB WORK

  [LAB](https://2021.igem.org/Team%3ANIT_Warangal/Lab)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANIT_Warangal/Experiments)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept)
  [RESULTS](https://2021.igem.org/Team%3ANIT_Warangal/Results)
  [SAFETY](https://2021.igem.org/Team%3ANIT_Warangal/Safety)
* ![](https://static.igem.org/mediawiki/2021/e/e9/T--NIT_Warangal--icons--NAVBARsocial.png)SOCIAL

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANIT_Warangal/Human_Practices)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANIT_Warangal/Collaborations)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANIT_Warangal/Contribution)
  [COMMUNICATION](https://2021.igem.org/Team%3ANIT_Warangal/Communication)
  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3ANIT_Warangal/Entrepreneurship)
* ![](https://static.igem.org/mediawiki/2021/b/b8/T--NIT_Warangal--icons--NAVBARtest.png)JUDGING

  [MEDALS](https://2021.igem.org/Team%3ANIT_Warangal/Medals)
  [JUDGING FORMS](https://igem.org/2021_Judging_Form?id=4077)

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At the beginning of the project, we were deeply intrigued by the
[engineering cycle](https://2021.igem.org/Engineering/Introduction)
and were keen on doing
multiple iterations in order to get the best result possible. But due to the ongoing pandemic, we were
restricted to doing just one cycle. The lack of computational resources and restrained access to the
laboratories hampered our project, but our motivation led us to complete the first iteration successfully, and
enabled us to learn more about the challenges we might face, and prompted us to devise suitable solutions to
overcome them. On this page, we will explain about how we proceeded with accomplishing the first iteration of
the engineering cycle, and the inspiration behind it.

## INSPIRATION

As the team members were exploring the iGEM website and wikis of other teams, the team was inspired by iGEM's
motto of "Local People solving Local Problems". Motivated by this goal, the team started finding out problems
that are faced by the local population and potential solutions to these problems. One problem that marred the
local community was that of harm to animals that were consuming cottonseed meal. After diving a bit more into
the problem we realized that the presence of gossypol was an inhibitor in widespread use of cotton seed meal or
cake for usage in the local community. We also realized the potential of using cottonseed as a protein source,
which can be proposed as a solution to solve widespread malnutrition problem across India and other developing
nations.

## RESEARCH

After an exhaustive study of existing methods for degossypolization of cotton seeds through various research
articles, we found four prominent methods which had succeeded in doing so. The methods we came across are as
follows:

* **Two stage solvent extraction process:** Utilizing aqueous and anhydrous acetone, a two stage solvent extraction
  has been developed which decreases free gossypol level in cottonseed and increases protein concentration to
  about 72.2% [1].
* **Treatment with ferrous sulphate:** This study showed that fish food containing 40% cottonseed meal when
  supplemented with 580 mg/kg Fe, considerably increased growth rate of fish. This was due to inactivation of
  free gossypol by iron[2].
* **Acidic solvent extraction:** Ethanol and acetone based solutions were used with phosphoric acid and water to
  hydrolyse the protein bound gossypol. Total gossypol level in cottonseed meal was reduced by
  5-10%[3].
* **Solid state fermentation:** A mixed fungal culture was prepared by mixing *Candida tropicalis* with
  *Saccharomyces cerevisiae*. After the solid state fermentation process, the cottonseed meal showed 79.5%
  detoxification rate
  for free gossypol and 59.5% for bound gossypol. 13.4% increase in crude protein was recorded in the fermented
  cottonseed cake[4].

However, we were not fully satisfied with this, and our zeal for innovation led us to think of a novel enzymatic
approach to degrade gossypol in a more efficient manner.

## DESIGN :

While designing the model of our project, we kept the expected final outcome in mind. The proposed functionality
of our model, which was to degrade gossypol, particularly influenced our thinking and aided us in designing the
intricacies of the enzymatic prototype.

Once our preliminary research regarding other approaches was completed in the 'Research Phase' of our project,
we set out to design our method of enzymatic degradation. For this, we had to decide 2 major components of our
project :

1. Expression organism that should be utilized to produce the enzyme
2. Media in which the enzyme will be produced

After a round of research and consulting with our PIs, it was determined that *Pichia pastoris* will be an ideal
candidate for expressing our enzyme.

*Pichia pastoris* is well known as a recombinant expression host system and is largely used for the commercial
production of various enzymes and therapeutic proteins. As a yeast, it shares the advantages of molecular and
genetic manipulations with *Saccharomyces*, and it has the added advantage of much higher heterologous protein
expression levels. Along with this, *Pichia pastoris* has the added advantage of showing several
post-translational modifications making it suitable for transformation. Cost effective production as well as easy
scalability make it suitable for large scale production[5].

Crude glycerol was identified as a prospective solution as it provides a reliable carbon source to the organism
to synthesize the enzyme. Crude glycerol is a low-value byproduct which is primarily obtained from the biodiesel
production process and since biodiesel production is predominant all across the world, it would be easily
available for a majority of the global stakeholders. Crude glycerol doesn't have any significant usage (until
it's purified) in other industries and thus is considered as waste, and so by utilizing crude glycerol we will
be recycling a waste product reducing environmental impact.

The enzyme selection process has been extensively discussed in the
[lab section](https://2021.igem.org/Team%3ANIT_Warangal/Lab).

pPIC9K is an expression vector for *Pichia* strains, which has a strong Alcohol oxidase promoter that helps in
higher protein expression and is identical to pPIC9 except for the presence of the kanamycin resistance gene for
in vivo screening of multiple copy inserts.

We used *Eco*RI and *Not*I restriction sites for double digestion and insertion of the *Melanocarpus albomyces*
laccase gene. The 2 sites are present in frame with the alpha-factor secretion signal.

Addition of the alpha-factor secretion signal to heterologous proteins in *Pichia pastoris* usually results in the
secretion of the intact heterologous protein to the extracellular medium [6].

![Gossypol image](https://static.igem.org/mediawiki/2021/a/a2/T--NIT_Warangal--imgs--engineeringsuccess--pPIC9k_laccase_plasmid.jpeg)

Fig 1: pPIC9k-Laccase plasmid [BBa\_K4077003](http://parts.igem.org/Part%3ABBa_K4077003)

## BUILD

Not having access to labs for the majority of the iGEM cycle made it difficult for the team to validate and
analyze the design of our project. A robust design which ensures a sustainable and feasible result for our
project was designed, along with incorporating aspects that will help us tweak certain components of the design
once we identify nuances that need to be addressed during the 'Learn' phase of the Engineering Cycle. A Brief
overview of this phase is as follows :

![Build info graphics](https://static.igem.org/mediawiki/2021/d/d1/T--NIT_Warangal--imgs--engineeringsuccess--build_flow_chart.jpeg)

Fig 2: Build phase flow chart

Once we identified our protein of interest,i.e., laccase we carried out propagation of the pPIC9k vector into
E.Coli DH5α which is a widely used and popular strain used for recombination.

The entire transformation process has been detailed in the [Proof of Concept](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept) section.

Once transformation is completed we screen for positive clones and further the phenotype of the organism will be
determined.

The two genes, *AOX*1 and *AOX*2 in *Pichia pastoris* code for alcohol oxidase. Majority of the alcohol oxidase
activity in the cell is accounted for by the *AOX*1 gene product. Expression of the *AOX*1 gene is induced to very
high levels by methanol and is tightly regulated. Typically more than 30% of the total soluble protein in cells
grown on methanol is accounted for by the *AOX*1 protein. While *AOX*2 is around 97% homologous to *AOX*1, the growth
on methanol is much slower than with *AOX*1.

Loss of the *AOX*1 gene, and for that reason a lack of maximum of the cell's alcohol oxidase activity, results in
a strain with a MutS (methanol utilization slow) phenotype. A MutS strain has a mutant aox1 locus, however is
wild-type for *AOX*2. It has decreased capacity to metabolize methanol and for that reason poor growth on methanol
medium[7].

Since we plan to produce laccase in a medium containing methanol it is important to select only Mut+(methanol utilization plus) plasmid.

## TESTING

![Gossypol image](https://static.igem.org/mediawiki/2021/5/50/T--NIT_Warangal--imgs--engineeringsuccess--testing_flow_chart.jpg)

Fig 3: Flow for testing phase

The recombinant plasmids would be used to produce laccase in crude glycerol as well as pure glycerol to compare
performance.

Laccase activity is determined by oxidation of the ABTS method[8]. ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) can be used to detect the binding of molecules to each
other. The absorbance of the ABTS+ cation can be used to evaluate the antioxidant capacity of drug solutions. It
is commonly used with blue multicopper oxidase enzymes, like laccases[9]. The non-phenolic dye ABTS is oxidized
by the laccase to form a more stable form of the cation radical. The concentration of the cation radicals can be
correlated to enzyme activity, and plays a major role in the intense blue-green color. It is observed to be at
420 nm.

After recording laccase activity, we proceeded to determine the protein concentration by the dye-binding method
of Bradford using BSA as standard.

Determination of the laccase activity will help us gain an idea of whether the strain is producing sufficient
amounts of laccase to degrade the gossypol.

## LEARN

The Learn Phase of the engineering cycle takes the outputs from the Test Phase and looks at how certain
parameters and factors could be changed in either the Design Phase or Build Phase or both that could lead to a
more efficient solution.

Information gained in this phase can be used to predict future scale-up procedures as we seek to make our
solution accessible and affordable to all stakeholders involved in the project. Here are some of the possible
challenges that we might encounter during the project:

**Possible Challenges[10]:*** Is the expression sufficient to produce enough laccase in both pure glycerol as well as crude glycerol used in the "Test Phase"?
* Is the final concentration of laccase after purification enough to carry out CSM treatment?
* Does the enzyme(laccase) function in the conditions predicted (temperature, pH, agitation, etc)?
* Is our laccase capable of removing enough gossypol from CSM for safe consumption ?
* When used by stakeholders in a practical scenario, does the laccase interact with other ligands and get used up before binding to gossypol?

Answering these questions will help us understand areas where our design perhaps needs to improve and can also provide
insight into aspects of our build which could be optimized.

## References :

* [1] Gerasimidis K, Fillou DT, Babatzimcpoulou M, Tassou K, Katsikas H. Preparation of an edible cottonseed
  protein concentrate and evaluation of its functional properties. Int J Food Sci Nutr. 2007 Sep;58(6):486-90.
  DOI: [10.1080/09637480701288488](https://doi.org/10.1080/09637480701288488). PMID: 17710592.
* [2] Gaber, M.M. & El-Halfawy, Magdy & Ramadan, Amal. (2012). Utilization of cottonseed meal supplemented with
  iron for detoxification of gossypol in Nile tilapia, broodstock and their impact on the hatchability of their
  progenies. Journal of Aquaculture Research and Development. 3.
  DOI: [10.4172/2155-9546.1000151](https://doi.org/10.4172/2155-9546.1000151).
* [3] Pelitire, Scott & Dowd, Michael & Cheng, Huai. (2014). Acidic solvent extraction of gossypol from
  cottonseed meal. Animal Feed Science and Technology. 195.
  DOI: [10.1016/j.anifeedsci.2014.06.005](https://doi.org/10.1016/j.anifeedsci.2014.06.005).
* [4] Vellaichamy, Mageshwaran & shaikh, A & Kathe, A. (2017). Optimization of process parameters for gossypol
  detoxification in chemical disinfected cottonseed cake by mixed fungal culture during solid state
  fermentation. Journal of scientific and industrial research. 76. 714-719.
* [5] Karbalaei, M., Rezaee, S. A., & Farsiani, H. (2020). *Pichia pastoris*: A highly successful expression
  system for optimal synthesis of heterologous proteins. *Journal of cellular physiology*, 235(9), 5867-5881.
  DOI: <https://doi.org/10.1002/jcp.29583>
* [6] Lin-Cereghino, G.P., Stark, C.M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., Agari, K., Moua, P.,
  Low, L.K., Tran, N., Huang, A.D., Nattestad, M., Oshiro, K.T., Chang, J.W., Chavan, A., Tsai, J.W. and
  Lin-Cereghino, J. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein
  expression in *Pichia pastoris*. Gene, [online] 519(2), pp.311-317.
   Available at:
  <http://www.sciencedirect.com/science/article/pii/S0378111913001765>
* [7]
  [Yeast Expression Support from Thermofisher](https://www.thermofisher.com/ae/en/home/technical-resources/technical-reference-library/protein-expression-support-center/yeast-expression-support/yeast-expression-support-getting-started.html)
* [8] Bourbonnais R, Leech D, Paice MG. Electrochemical analysis of the interactions of laccase mediators with
  lignin model compounds. *Biochimica et Biophysica Acta*. 1998;1379(3):381-390.
  [[PubMed](https://www.ncbi.nlm.nih.gov/pubmed/9545600)]
  [[Google Scholar](https://scholar.google.com/scholar_lookup?journal=Biochimica+et+Biophysica+Acta&title=Electrochemical+analysis+of+the+interactions+of+laccase+mediators+with+lignin+model+compounds&author=R+Bourbonnais&author=D+Leech&author=MG+Paice&volume=1379&issue=3&publication_year=1998&pages=381-390&pmid=9545600&)]
* [9] Milena Morandi Vuodslo, ... Mário Roberto Maróstica Junior, in
  [Bioactive Compounds](https://www.sciencedirect.com/book/9780128147740/bioactive-compounds),
  2019
* [10] Inspired by [https://2020.igem.org/Team:ULaval/Engineering](https://2020.igem.org/Team%3AULaval/Engineering)

# Sponsors

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---

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iGEM NIT Warangal

 National Institute of Technology, Warangal

 Warangal, Telangana, INDIA - 506004

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# Team:NIT Warangal/Experiments

iGEM NIT\_Warangal

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* [![](https://static.igem.org/mediawiki/2021/5/5e/T--NIT_Warangal--icons--NAVBARhome.png)
  HOME](https://2021.igem.org/Team%3ANIT_Warangal)
* ![](https://static.igem.org/mediawiki/2021/7/7a/T--NIT_Warangal--icons--NAVBARgroup.png)PEOPLE

  [TEAM](https://2021.igem.org/Team%3ANIT_Warangal/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANIT_Warangal/Attributions)
  [SPONSORS](https://2021.igem.org/Team%3ANIT_Warangal/Sponsors)
* ![](https://static.igem.org/mediawiki/2021/7/73/T--NIT_Warangal--icons--NAVBARpresentation.png)PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3ANIT_Warangal/Description)
  [ENGINEERING](https://2021.igem.org/Team%3ANIT_Warangal/Engineering)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANIT_Warangal/Implementation)
  [PARTS](https://2021.igem.org/Team%3ANIT_Warangal/Parts)
  [NOTEBOOK](https://2021.igem.org/Team%3ANIT_Warangal/Notebook)
* ![](https://static.igem.org/mediawiki/2021/f/f1/T--NIT_Warangal--icons--NAVBARlab.png)LAB WORK

  [LAB](https://2021.igem.org/Team%3ANIT_Warangal/Lab)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANIT_Warangal/Experiments)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept)
  [RESULTS](https://2021.igem.org/Team%3ANIT_Warangal/Results)
  [SAFETY](https://2021.igem.org/Team%3ANIT_Warangal/Safety)
* ![](https://static.igem.org/mediawiki/2021/e/e9/T--NIT_Warangal--icons--NAVBARsocial.png)SOCIAL

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANIT_Warangal/Human_Practices)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANIT_Warangal/Collaborations)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANIT_Warangal/Contribution)
  [COMMUNICATION](https://2021.igem.org/Team%3ANIT_Warangal/Communication)
  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3ANIT_Warangal/Entrepreneurship)
* ![](https://static.igem.org/mediawiki/2021/b/b8/T--NIT_Warangal--icons--NAVBARtest.png)JUDGING

  [MEDALS](https://2021.igem.org/Team%3ANIT_Warangal/Medals)
  [JUDGING FORMS](https://igem.org/2021_Judging_Form?id=4077)

![top-overlayed-image](https://static.igem.org/mediawiki/2021/d/dc/T--NIT_Warangal--icons--vectorsGraphics--experiments_top.jpeg)

Laboratory experiments are not only important in bringing a theoretical project to reality at the ground level,
but also in verifying if the computational models work in the natural scenarios. We went ahead with the
experiments and tried to do all the required experiments for our projects in the limited exposure that we got to
the laboratories of our institute. The protocols of the experiments that we conducted can be found below:

## Experiments

# Sponsors

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---

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iGEM NIT Warangal

 National Institute of Technology, Warangal

 Warangal, Telangana, INDIA - 506004

# Connect with us

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# Team:NIT Warangal/Results

iGEM NIT\_Warangal

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* [![](https://static.igem.org/mediawiki/2021/5/5e/T--NIT_Warangal--icons--NAVBARhome.png)
  HOME](https://2021.igem.org/Team%3ANIT_Warangal)
* ![](https://static.igem.org/mediawiki/2021/7/7a/T--NIT_Warangal--icons--NAVBARgroup.png)PEOPLE

  [TEAM](https://2021.igem.org/Team%3ANIT_Warangal/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANIT_Warangal/Attributions)
  [SPONSORS](https://2021.igem.org/Team%3ANIT_Warangal/Sponsors)
* ![](https://static.igem.org/mediawiki/2021/7/73/T--NIT_Warangal--icons--NAVBARpresentation.png)PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3ANIT_Warangal/Description)
  [ENGINEERING](https://2021.igem.org/Team%3ANIT_Warangal/Engineering)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANIT_Warangal/Implementation)
  [PARTS](https://2021.igem.org/Team%3ANIT_Warangal/Parts)
  [NOTEBOOK](https://2021.igem.org/Team%3ANIT_Warangal/Notebook)
* ![](https://static.igem.org/mediawiki/2021/f/f1/T--NIT_Warangal--icons--NAVBARlab.png)LAB WORK

  [LAB](https://2021.igem.org/Team%3ANIT_Warangal/Lab)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANIT_Warangal/Experiments)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept)
  [RESULTS](https://2021.igem.org/Team%3ANIT_Warangal/Results)
  [SAFETY](https://2021.igem.org/Team%3ANIT_Warangal/Safety)
* ![](https://static.igem.org/mediawiki/2021/e/e9/T--NIT_Warangal--icons--NAVBARsocial.png)SOCIAL

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANIT_Warangal/Human_Practices)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANIT_Warangal/Collaborations)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANIT_Warangal/Contribution)
  [COMMUNICATION](https://2021.igem.org/Team%3ANIT_Warangal/Communication)
  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3ANIT_Warangal/Entrepreneurship)
* ![](https://static.igem.org/mediawiki/2021/b/b8/T--NIT_Warangal--icons--NAVBARtest.png)JUDGING

  [MEDALS](https://2021.igem.org/Team%3ANIT_Warangal/Medals)
  [JUDGING FORMS](https://igem.org/2021_Judging_Form?id=4077)

![top-overlayed-image](https://static.igem.org/mediawiki/2021/2/22/T--NIT_Warangal--icons--vectorsGraphics--results_top.jpeg)

Owing to the ongoing pandemic, our institute remained closed till the month of October, thus not giving us a
good amount of time and exposure to the laboratories for the wet lab part of our project. The detailed protocols
and experiments performed can be found on the
 [Experiments](https://2021.igem.org/Team%3ANIT_Warangal/Experiments) and
[Proof of Concept](https://2021.igem.org/Team%3ANIT_Warangal/Proof_Of_Concept) pages. With only two members of
our team allowed on campus for a short amount of time, the following results were obtained:

### Our results

Since we had access to the labs for only one week, we were unable to carry out laccase treatment on cottonseed meal (CSM) before
wiki freeze. We plan to present the CSM treatment results during our judging session as by then we will have
sufficient time to conduct the required experiments.

In our preliminary studies of Cottonseed Meal treatment by laccase, Gossypol degradation was found to be **72%**.
However, we will be conducting replications to confirm the results of the experiments and also perform optimizations as currently the treatment is not optimized.

# Sponsors

[![etl_logo](https://static.igem.org/mediawiki/2021/5/5a/T--NIT_Warangal--common--sponsorsLogos--etl_logo.png)](https://emmessartech.org/)

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---

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iGEM NIT Warangal

 National Institute of Technology, Warangal

 Warangal, Telangana, INDIA - 506004

# Connect with us

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Icons made by
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