Glutamine Synthetase 2

Student Name: Navya Rawal

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DVHS Accelerated Biotechnology and Research

Waksman Student Scholars Program -- 2023-2024

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Scientific Presentation: WSSP

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Abstract

The purpose of the DNA Sequence Analysis WSSP is to identify proteins that are present in Landoltia punctata through wet labs and bioinformatics. Landoltia punctata, better known as Duckweed, is a highly researched plant because of its ability to be used in environmentally sustainable products. Samples from Rutgers University were analyzed through overnight cultures, PCR, plasmid miniprep, restriction digest, and gel electrophoresis. Although there were some errors with the unknown samples and the original sample was unreadable, the new orphan clone had defined peaks and minimal errors. The name of the protein was identified through general searches of the Basic Local Alignment Search Tool (BLAST). With the most likely reading frame from BLASTx, the Open Reading Frame (ORF) of the DNA clone was determined by the ORF Toolbox with the nucleotide sequence. Based on these searches we were able to determine the protein was the Glutamine Synthetase. A thorough literature analysis was completed on the protein sample. Using the amino acid sequence established by the ORF, the potential protein was compared to databases of homo sapiens’ proteins, the Protein Data Bank (PDB), Transcriptome Shotgun Assembly (TSA) for Landoltia Punctata, and TAIR database for Arabidopsis thaliana to find its function and structure. Gene expression was found through the Arabidopsis eFP Dataset and Cell eFP Browser with the Arabidopsis homolog. This protein is key to plant growth which is why it is highly expressed within the chloroplast and mitochondria. It interacts with 9 other proteins and is present in plant cells, a similar protein exists in human cells. The protein is consistently expressed through majority of the different biotic and abiotic stresses. The WSSP website was my primary source of information, along with NCBI BLAST tools.

DUCKWEED

Landoltia Punctata

Kingdom Plantae Phylum Tracheophyta Class Liliopsida Order Alismatales Family Araceae Genus Spirodela Schleid

Found in the serene waters of North America, the tranquil ponds of Europe, and the lush waterways of Asia. This versatile plant can thrive in a wide range of environments.

Landoltia Punctata is a diminutive aquatic plant of immense scientific significance. This plant is characterized by its miniature size, floating habit, rapid reproductive capacity, simple structure, and global distribution, thriving in diverse aquatic ecosystems. Notably, duckweed’s ability to efficiently absorb nutrients from its surroundings, coupled with its protein-rich composition, has sparked interest across various scientific domains, including biotechnology, agriculture, and pharmaceutical research.

Duckweed's simple genetic structure, small genome size, and rapid growth make it an accessible and valuable candidate for genetic research. Researchers have conducted DNA sequencing to unravel its genetic code, revealing genes responsible for various traits. Moreover, duckweed's high protein content, reaching up to 40% on a dry weight basis, holds promise for applications in animal feed and sustainable biofuel production. Understanding the genes and proteins involved in nutrient absorption is pivotal for its role in wastewater treatment. Furthermore, duckweed's genetic insights open doors to pharmaceutical research, exploring its potential in producing bioactive compounds for drug discovery and development, underscoring its significance in diverse scientific disciplines. Overall, Duckweed research offers a myriad of applications with far-reaching benefits. It plays a pivotal role in the production of sustainable biofuels, aids in wastewater treatment to purify our water sources, removes excess nutrients from aquatic ecosystems, serves as a nutritious animal feed, and contributes to groundbreaking pharmaceutical research.

Methods

Overnight Culture

PCR

Gel Electrophoresis

Plasmid

Miniprep

Restriction

Digest

Select white bacterial colonies that contain the CDNA inserts to set up liquid cultures which are grown to liquid saturation

Amplifies fragment of DNA that contains the insert through cycles of denaturing, annealing, and elongation

Run an agarose gel to determine the size of the PCR fragment and the CDNA insert to determine if the insert is large enough to sequence and equal to one another

Digest purified plasmid to verifies the size of the cdna insert and considers quality of the insert

Purify plasmid DNA away from the bacteria, genomic DNA, RNA, and proteins.

PCR and RDG Mock Up Results Based on 65NR1.23

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65NR1.23 was sent to be sequenced because Uncut and Cut did show up. The PCR band did not show up because of issue in TAQ Polymerase, master mix, or dNTPs. However when the sequencing results returned, the waveform was unreadable. There were several peaks at each bp, indicating that either the template concentration was too low resulting in the signal intensity is of borderline readability and data got detected by the software only some of the time or bad primer was used.

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DNA sequence analysis was based on clone 2031501.20

Waveform: Unedited

Waveform: Edited

Waveform Information

Length: 646 bp

Start: A57

Quality: Partial

End: A703

BLASTn

nr/nt database

est database

BLASTn Analysis

The edited sequence is significantly similar to sequences found in other organisms. However it is not significantly similar to the sequences found in different kingdoms, it is found in just Mesangiospermae or flowering plants. Additionally, majority of the results have a very similar Query End.

BLASTx

standard database

BLASTx Analysis

The edited sequence is significantly similar to sequences found in other organisms. However it is not significantly similar to the sequences found in different kingdoms, it is found in just Mesangiospermae or flowering plants. Additionally, majority of the results have a similar Query starts and ends.

ORF & Analysis

Using the results from BlastX, Frame +1 would best code for a protein sequence. The ORF starts at A1 and ends at C643.

Protein Sequence

BLASTp

standard database

BLASTp Analysis

The sequence does contain, but does not start with a start codon. However a stop codon is not present in the sequence. This indicates that the sequence is a partial.

BLASTx vs BLASTp Analysis

The BLASTx and the BLASTp search resulted in the same proteins with similar E-values. The start position for all of the results were the same, however BLASTx and BLASTp had different stop positions.

BLASTx

BLASTp

BLAST Summary

Based on the results provided from BLASTx, BLASTn, BLASTp and ORF, the protein sequence is most likely a partial without a 5’ UTR.

Analysis Question 1

A) What is the FULL name of your protein? What is the function of your protein? List the sites where you found this information. (books, Google, Wikipedia, PubMed)

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Glutamine Synthetase, (CDD - Conserved Domain Database) glutamine synthetase catalyzes the condensation of glutamate and ammonia to form glutamine, (NIH - National Library of Medicine) Glutamine synthetase (GS) is the adenosine triphosphate (ATP)-dependent enzyme that catalyses the synthesis of glutamine by condensing ammonium to glutamate. In the circulatory system, glutamine carries ammonia from muscle and brain to the kidney and liver. In brain reduction of GS activity has been suggested as a mechanism mediating neurotoxicity in neurodegenerative disorders. In cancer, the delicate balance between glutamine synthesis and catabolism is a critical event.

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B)Perform a BLASTP of Homo sapiens (taxis 9606) to determine if there is a similar protein in humans. If there is a homolog, indicate the:

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i) The Accession number of the protein: AAI27884.1

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ii) The Protein name: GLUL protein, partial

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iii) What does this tell you about the evolution of this protein?

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Because the BLASTp search resulted in a 55% Identity and 73% Positive, it indicates that there are somewhat similar proteins in humans. Our protein has some parts of it that are conserved through evolution, indicating it plays an important role in cell function.

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Analysis Question 2

Size of Sequence based on DNA Sequence Analysis: 647 bp

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The clone being analyzed was not prepared by me, so the difference is N/A.

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Analysis Question 3

Search the PDB with your protein sequence to determine if there is a homologous protein in which the structure has been determined.

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a) The PDB accession number (4 digits) for the structure: 7V4I

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b) Which organism is it from: Camellia sinensis

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c) Perform a BLASTP 2 Sequences alignment between your protein and the PDB protein. What is the % Identity AND % Positive?: % Identity: 83% (178/215) & % Positives: 89% (192/215)

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d) How many alpha helices and beta-strands are in the protein: 12 alpha helices and 12 beta-strands

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e) Are the beta strands are parallel, antiparallel or both: Both parallel and antiparallel strands are present in the structure of the protein.

Analysis Question 4

Perform a BLASTN search of the Transcriptome Shotgun Assembly (TSA) database that is limited to Landoltia punctata.

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A) Examine the search results and enter in the sequence ID of the best contig match and the % sequence identity between the two sequences and the total number of base pairs of the entire contig sequence (not just the region that matches).

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A. Sequence ID: GAQZ01055857.1, Identities: 645/647(99%), Length: 2008 bp

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B) Download the TSA contig DNA sequence and perform a BLASTX search with the sequence. Does the contig sequence match a protein? If so, indicate the protein Accession number, the name of the protein and the E-value of the match.

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B. chloroplast glutamine synthetase, Sequence ID: UJI64966.1, E-Value: 0.0

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Analysis Question 5

Perform a BLASTP search of the TAIR database. Use BioGrid to find interactors

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1) What is the name of the Gene locus you used in the search of the BioGRID database: AT5G35630

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2) How many different proteins interact with this protein? 9 interactors

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3) Name one of the proteins (AT number) that interacts with your protein that is biologically significant. Indicate its function.

• AT1G80670: Rae1-like protein
• AT3G14980: histone H3 acetyltransferase IDM1
• AT4G30840: transducin/WD40 domain-containing protein

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Analysis Question 6

During Development, glutamine synthetase is highly expressed in leaf development during stages 1 through 12 and flower development during stages 12 and 15.

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Localization of protein in the cell is highly expressed in chloroplast. Medium expression in the mitochondria. Minimal expression in the cytosol, vacuole, golgi, plasma membrane, and cell wall.

For tissues, the protein is highly expressed in mesophyll cells, top of the stem, ovary tissue, rib meristem, and S17.

Analysis Question 6 Continued

Abiotic Stresses: Less expression with Osmotic (300 mM Mannitol) and Salt (150mM NaCl) after 6, 12, 24 hours. However, the protein is generally always highly expressed through other abiotic stresses.

Biotic Stresses: Less expression after 18 and 48 hours after exposure to Botrytis cinerea and after 24 hours of exposure to Infiltrating Pseudomonas syringae for both virulent and avirulent. Continuous high expression throughout other biotic stresses.

Analysis Question 7

Utilize gene editing tools such as CRISPR/Cas9 to generate GS knockout or overexpression mutants in Landoltia.

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Analyze the phenotypic effects of these modifications on growth, nitrogen assimilation, and stress responses.

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Expected results: Knockout mutants might exhibit impaired growth or altered nitrogen utilization compared to wild-type plants, whereas overexpression mutants may show enhanced growth or nitrogen assimilation capacities.

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Acknowledgements

Thank you to the following:

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Andrew Vershon (WSSP)- WSSP/ DSAP

John Brick (WSSP)- Reviewed DSAP

Janet Mead(WSSP)- Clones and DSAP

Hilary Dito and Azine Davouzedeh( CCC ROP)- DNA Sequencing and Reagents

Katherine Huang(DVHS)- High school Instructor

Lab partners (DVHS)- Aanya L and Msgana M

Thank you to everyone at Rutgers for providing students this opportunity. Thank you to Ms.Huang for mentoring me throughout this process and teaching me about biotechnology. Thank you to my peers and lab partners for supporting me throughout this process.

Thank You

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