Team:MRIIRS FARIDABAD/Poster

MRIIRS_FARIDABAD Li-Koff

Application No to the design application is 202011034154

To Detect and Degrade N-Nitrosamines

Authors: Shivani Khare¹, Sristhi Chopra², Hardik Grover¹, Tarini Vohra¹, Umra Mahmood¹, Kartik Jatwani¹, Dewashish Kaushik¹, Abhilansh Pandey¹, Shivam Bhardwaj¹, Alokit Tanwar¹, Japneet Singh¹, Chander Shekhar¹, Mrugank Dake⁵, Kannan Krishnamoorthy³, Kapila Kumar⁴

¹ Team Member, iGEM team of MRIIRS, Faridabad
² Student Leader, iGEM team of MRIIRS, Faridabad
³ Instructor, iGEM team of MRIIRS, Faridabad
⁴ Primary PI, iGEM team of MRIIRS, Faridabad
⁵ Advisor (Ex-iGEM), IISER Tirupati

Abstract:

N-Nitrosamines are group of alkylating compounds formed from the reaction between nitrites, secondary and tertiary amines. Majority of these compounds are known to be potent carcinogens. They are found in pharmaceuticals, cosmetics, processed meats, alcoholic beverages, cigarette smoke, suspended particulate matter (SPM) etc. But, their presence in potable water post disinfection stage in water treatment plants is most alarming; as their formation depends upon temperature and pH and not on the precursor molecules forming them. Quantifying them through conventional techniques of LC/MS, GC/MS, HPLC, etc., is time consuming, labour intensive and a costly affair. Hence, li-koff pitches in as an alternate SynBio solution for an easy detection and estimation of these alkylating compounds in water samples. Further, it also degrades some of these compounds upon detection.

Inspiration

Carbon dioxide is often considered to be the principal contributor to global warming. Not entirely wrong, but the presumption that carbon pollution is the greatest cause of climate change is, in fact, erroneous.
Nitrogen pollution is currently more concerning owing to the dearth of awareness and research on it.
It was this realization that led to the inception of our project and motivated us to develop a bio-sensing solution by exploiting their alkylating property.
The detrimental potential of the alkylating agents like nitrosamines includes their carcinogenicity and contamination of the environment including water resources that inspired us to design a bio-sensing solution to detect these.
Our bacterial cell-based bio-sensing solution is based on ADA Regulon that is involved in a natural adaptive response mechanism in E.coli.
It gets activated by lesions formed due to the DNA damaging effect of alkylating agents like Nitrosamines and subsequently repairs it.

Project Description and Mechanism

The primary goal of our project is to engineer E.coli that detects the presence of alkylating agents like nitrosamines in drinking water:

For this, we have constitutively expressed the ada response protein which is released in E. coli upon DNA damage caused due to alkylation. Its constitutive expression allows us to report the slightest concentration of nitrosamines present in water sample. This Ada protein undergoes methylation and further activates the synthesis of the reporter protein present downstream to the ADA-AlkB promoter showing the presence of the targeted compounds via fluorescence.

We also propose to specifically degrade NDMA:

Pseudomonas mendocina KR1 possesses the T4 monooxygenase gene which has the ability to degrade NDMA (Sharp et al, 2005). We will introduce this gene in E. coli which, in the presence of toluene, would get activated by methylated Ada protein.

Mechanism:

E.coli has a natural adaptive response mechanism known as the ADA response. It is a DNA damage repair mechanism that gets induced by an O6-methylguanine (O6-MeG) lesion formed due to methylation of DNA in the presence of alkylating agents like nitrosamines (Watson & McGregor, 2010). This damage caused in DNA due to methylation is repaired using four Ada response proteins, encoded by three different operons of the “Ada Regulon” (Mielecki & Grzesiuk, 2014). The methyltransferase activity of Ada response protein aids in a methyl shift reaction from O6-MeG lesion to the Ada protein. For the purpose of our project, we have selected the ADA-AlkB operon which gets induced by methylated Ada, which acts as a transcriptional activator, in the presence of an O6- methylguanine lesion. The Ada protein, upon methylation, will further activate the synthesis of the reporter protein which is placed downstream to an Ada induced promoter (Ada- AlkB promoter) hence, showing the presence of the targeted compounds via fluorescence.

Engineering (Workflow)

For preliminary testing, we placed an EGFP reporter gene with terminator downstream to an Ada-induced promoter and cloned it in pCMV-6-AC-His plasmid.
This was to be followed by artificial spiking of water samples to conduct assay experiments.
Lockdown due to Covid-19
Hence, we transitioned to in-silico and with the use of softwares like benchling, we completed the designing of the general testing bio-brick and checked it using virtual digest.

We would like to thank Dr Nandula Raghuram for providing us with the primers for gene amplification in preliminary testing experiments.
Constitutive expression of ada (biobrick) will increase the initial concentration of ada which happens to be quite low in the natural system.
The idea of using constitutive promoter for increasing initial concentration of Ada response protein is one of the valuable contributions of Dr Siddharth Manvati in designing the General Testing Biobrick (detection).

Contribution (Parts)

ADA-AlkB gene optimized sequence (Registered as BBa_K3587000)

This is a basic part registered by our group on iGEM repository. This part basically belongs to the genome of E.coli which forms the DNA repair elements and code for Ada methyltransferase and AlkB dioxygenase. The sequence used is optimized using various tools in order to accommodate for restriction sites compatible with standard assembly.

Ada methyltransferase is involved in a methyl-shift reaction that takes place at the site of lesion formed. The methylated form of Ada protein exists in an irreversible suicidal state which is involved in transcriptional activation of the promoters of the ADA regulon (Lindahl et al., 1988).

E. coli AlkB-encoded AlkB (EcAlkB) protein belongs to the dioxygenase superfamily which is involved in oxidative demethylation of NA bases. Additionally, It requires non-heme Fe2+ and cofactors, 2-oxoglutarate (2OG) and oxygen (O2). This reaction type is involved in restoring the native base conformation in the nucleic acids (Aravind & Koonin, 2001).

ADA-AlkB Promoter + RBS (Registered as BBa_K3587005)

It is an efficient promoter of ADA regulon with a Ribosomal binding site (RBS). This promoter exhibits about a 10-fold higher affinity for the Ada protein (Landini & Volkert, 1995) than the other promoters of the regulon. The reporter sequence is placed downstream to this promoter.

This sequence comprises 10-15 bp stretches where the protein DNA interaction between methylated Ada and nucleic acid sequence takes place in order to act as a transcriptional activator for the subsequent downstream gene.

Proposed Implementation and Future Work

In future, we propose to use the parts of general testing biobrick along with T4-MO gene derived from Pseudomonas mendocina KR 1 in order to detect and selectively degrade one of the dangerous alkylating nitrosamine- NDMA.
The thesis of Dr Christopher Sales, University of Drexel, Pennsylvania validated the use of T4-MO for degrading NDMA.
We propose to create a biosensor, in the form of a bio-sensing solution, that can detect the presence of these alkylating agents.
Practical validation of this bio-sensing solution can help us to transform it into dipsticks and paper-based kits ultimately providing a substitute for their expensive detection.
These kits would be quite advantageous in lieu of their ease of handling as they do not require any trained personnel for its use.
The feedback from Delhi Jal Board, India will help us to design experiments for practical validation of the biosensing solution.
The feedback from Dr Satyakam Patnaik, scientist Indian Institute of Toxicology and Research, Lucknow, India has helped us in proposing the future application of our biosensing solution.

References:

Uphoff S, Lord ND, Okumus B, Potvin-Trottier L, Sherratt DJ, Paulsson J (2016). "Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation." Science 351(6277);1094-7. PMID: 26941321 Lee YM et al., 2015. RPM peptide conjugated bioreducible polyethylenimine targeting invasive colon cancer.

Landini P. Volkert MR. Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein. Evidence for a new consensus sequence for Ada-binding sites. J Biol Chem. 1995;270:8285–8289.

The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Aravind L, Koonin EV Genome Biol. 2001; 2(3):RESEARCH0007.

Review Regulation and expression of the adaptive response to alkylating agents. Lindahl T, Sedgwick B, Sekiguchi M, Nakabeppu Y Annu Rev Biochem. 1988; 57():133-57.

Sales, C. M. (2012). Functional genomics of the bacterial degradation of the emerging water contaminants: 1,4-dioxane and N-nitrosodimethylamine (NDMA). UC Berkeley.

Priti Mulchandani, Wilfred Chen, Ashok Mulchandani, Joseph Wang, Liang Chen, Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase, Biosensors and Bioelectronics, Volume 16, Issues 7–8, 2001, Pages 433-437, ISSN 0956-5663,

Results

Project Modelling Part 1

Mathematical model is based on ordinary differential equations that simulate the working of our modified cell based bio-sensing solution. This will also help us to adjudge feasibility of the designed experiment to modify E.coli. This will help us in optimization of our system and will assist in result interpretation.

Main Objectives are:

To engineer E.coli cell that can detect the presence of alkylating agents like nitrosamines in water.
To predict the sensitivity and limit of detection of our cell based bio-sensing solution.

Equations:
Following ordinary differential equations were designed keeping above parameters in consideration:

ƀ_d Ǭ_m f(E) - ϒ_mM     - Eq.1

β_I - ϒ_iI     - Eq.2

Q(E)= Ǭ_v= Ǭ_m f(E)     - Eq.3

Parameters:
The following parameters were used to design and construct Ordinary Differential Equation:

The DNA damaging effect of alkylating agents like nitrosamines is characterized by the formation of O6-MeG or O4-MeG lesions.
The Ada response protein of ADA regulon, one of the proteins involved in Adaptive response for DNA repair, scans the DNA and gets methylated when comes in contact with O6-MeG or O4-MeG lesion.
The methylated Ada protein acts as a transcriptional activator of three operons of ADA regulon. One of the operon has gene for Ada protein which leads to increase in the number of mRNA coding for Ada response protein.
First equation is to find the rate of mRNA (mRNA of Ada protein) formation in E.coli when Ada responds to alkylated DNA. As per equation 1, the rate of mRNA can be defined as difference between the rate production of mRNA and rate of degradation of mRNA.
The rate of Ada mRNA production, corresponding to the repair mechanism, is represented as
ƀ_d *Ǭ_m *f(E)

and the rate of mRNA degradation as

ϒ_m*M

Therefore, the final equation rate of mRNA production becomes

ƀ_d *Ǭ_m *f(E) - ϒ_m*M (Equation 1)

Project Modelling Part 2

In our system we have placed fluorescent reporter gene (GFP) downstream to a methylated Ada induced promoter. Therefore, the expression of the fluorescence reporter (GFP) will confirm the presence of alkylating agents like Nitrosamines.
Equation 2 demonstrates the rate of GFP expression, (i.e. ),

β_I *M

is the protein that is translated by DNA and the degradation of that protein is represented as

ϒ_I *I

Therefore, the final equation for rate of GFP expression becomes

β_IM- ϒ_II (Equation 2)
As per the mechanism of Ada response protein, the Ada can transcriptionally activate the operons of Ada regulon in methylated form (active form) only.
When the unmethylated form (inactive form) of Ada comes in contact with the lesion, it gets converted to an active methylated form due to the methyl shift reaction taking place at the point of contact.
Therefore,

Q(E)= Ǭ_v= Ǭ_m f(E) (Equation 3)

Graph MATLAB Code:

function u=vdpol2(t,x)
u=[3000 - 3.3*x(1) , 20*x(1)-0.4*x(2)]';
x0=[0.1 1];
t0=0; tf=120;
tic
[tout, xout]=ode23('vdpol2', [t0, tf], x0);
toc
hp=plot(tout, xout(:,1),tout, xout(:,2));
xlabel('Time(seconds)');

Graph:

Collaboration

Webinar was conducted in collaboration with iGEM_MSP with 4th&5th standard students of Manav Rachna International School, Faridabad, National Capital Region.

Webinar was conducted in collaboration with iGEM_IISER Tirupati with 11th&12th standard students of Manav Rachna International School, Faridabad, National Capital Region.

Syn Bio 101; Video series was curated to help learners understand synthetic biology concepts. These videos were posted on the YouTube channels of both the teams; igem_manavrachna and iGEM IISER Bhopal

We also collaborated with three other iGEM teams to help spread synthetic biology projects.

Human Practice

Webinar:
Two different series of webinars were conducted:
Bio-Building blocks-webinars (five) were conducted with school students of 6-12th to explain them about iGEM, Synthetic Biology and Li-Koff.
Saving Environment through innovation- webinars (seven) were conducted with non-science students (school+college; undergraduate). These webinars focussed on ‘Environment Consciousness’.

NGO:
Visited several clusters of ROBINHOOD Army, New Delhi.
Taught students about basic biology concepts like microbial degradation.
The students were taught about the importance of sanitisation in pandemic time.

Planters:
Earthen planters were designed containing all information about our project.
These were then distributed to the general public to help them understand the existing problem and solution we provide.

Li- Koff Special Comic:
We designed a comic in both Hindi and English language. This was distributed through both online and offline (150 copies) mode to several students.

Media:
Radio show was conducted with Radio Manav Rachna 107.8 FM local Faridabad radio station with 1.6 million listeners to spread awareness about our project.
Our project story was also covered by prominent print media houses in India: The Hans Times, The Times of India and The Better India.

SynBio101 - Video series:
Curiosity and zeal of students to know about synthetic biology during webinars led us to create synbio 101 in collaboration with IISER Bhopal. Synbio 101 is a video series on synbio and its applications.

Spreading Synthetic Biology:
Collaborated with three different iGEM teams:

iGEM UULM
iGEM RUM
iGEM Stanford University

Presented their promotional videos from our YouTube channel iGEM_ManavRachna to spread synthetic biology projects.

Science Communication

NGO visits/Social awareness:

Planters - We informed and interacted with the general public by distributing self-designed earthen planters. These were potted with Peace Lily plant which helps in reducing Nitrogen Pollution.
NGO - We went on to visit the students of Robin Hood Army, New Delhi. The interaction with these students was fruitful as we could make them understand many biological processes.

Webinars:
Through our two different series of webinars we could interact with many people belonging to the life science community and outside the community.
The webinars were conducted on both National and International platforms.
Starting from interacting with students of 4th standard to interacting with post graduate students we covered students of almost all age groups.

Multimedia:

Li-Koff Special Comic - Comic was designed to help people easily understand the concept of synbio and our project. This was distributed to various people through online and offline mode.
Radio Show - A radio show was conducted on Radio Manav Rachna 107.8 FM local Faridabad radio station.
'The Times of India', 'The Hans Times' and 'The Better India' - Prominent print media houses of India covered our project stories. This helped us to reach a wider audience.

Idea Presentation:

Youth Jalsa
Microvitae
Envisage, Khalsa college, Delhi University

Syn Bio 101:
We created a series of videos on synthetic biology and published them on our YouTube channel. This was done in collaboration with IISER Bhopal.

Integrated Human Practice

Dr. Siddharth Manvati: Research Scholar at Jawaharlal Nehru University and Co-founder of ClearMeat, India's first lab-grown meat company.

He gave us many useful insights such as adding a constitutive ada promoter and helped us in designing basic biobrick.
Prof. N. Raghuram: Designation: Chair Professor and Former Dean, School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi.

Helped in understanding the parameters to base our biosensing solution on.
Provided us with required primers as a keen gesture to help us build the idea.
Dr. Satyakam Paitnaik: Designation: Scientist at Indian Institute of Toxicology and Research (IITR), Lucknow.

Helped us in designing the proposed implementation of our project and ease of use of applications outside the lab in the real world situation.
Dr. J.S Dheer: Designation: State Programme Officer, DGHS, Govt. of Delhi.

His comments helped us in designing the mathematical stance of our project.
And he also thinks that this project will prove a milestone in preventing various Nitrosamine associated carcinogenic and other pathological conditions among the population.
Vijay Pal Singh: Designation: Joint Director at FSSAI, New Delhi

His views about nitrosamines, the way industry deals in handling such compounds and bringing small but significant changes to regulate their levels brought us to think of ease of application of our project.
Mr. Ashutosh Kaushik: Designation: Director, Water Treatment Quality Department, Delhi Jal Board

It was here we learnt how our project should be applicable and how it should benefit our targeted audience potential end users.
Dr. M C Gupta: Senior Scientist at Rasi Seeds Pvt. Ltd

He complimented that this product can definitely help in reducing Nitrosamines percentage in the environment.

ACKNOWLEDGEMENTS/SPONSORS/DISCLAMIER