iGEM 2025 — Team ict-mumbai

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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# Notebook

#### Protocol, Design, Verification

---

Our journey with IGEM, from initial ideation to building a model of our project. Capturing our progress, setbacks, and learning curves that have helped shape our project and acrry it forward to becoming a reality. A record of not just the growth of our project, but also of the individuals working tirelessly on the project.

![page-centrepiece](https://static.igem.wiki/teams/5893/pagelogo/labnotebook.webp)

[Construction and Characterization of GAT1 Knockout in S. cerevisiae](#section1)
[Week 1: Primer Design and Preparation](#section1-1)
[Week 2: Disruption Cassette Construction & Yeast Transformation](#section1-2)
[Week 3: Verification of GAT1 Knockout](#section1-3)
[Week 4: URA3 Marker Excision](#section1-4)
[Week 5: Phenotypic Testing – Nitrogen Analysis](#section1-5)
[Enhancement of Nitrogen Assimilation in ΔGAT1 S. cerevisiae via GDH1 and GLN1 Overexpression](#section2)
[Week 1: Target Selection and Experimental Design](#section2-1)
[Week 2: Plasmid Construction and Yeast Transformation](#section2-2)
[Week 3: Culture Inoculation and Expression Verification](#section2-3)
[Week 4: Nitrogen and Protein Analysis](#section2-4)
[Engineering S. cerevisiae for Nitrogen Recycling and Preliminary Assessment of Enhanced Nitrogen Assimilation](#section3)
[Week 1: Engineering Yeast for Nitrogen Recycling](#section3-1)
[Week 2: Preliminary Assessment of Nitrogen Assimilation](#section3-2)
[Development of Melanin–PVA Sheets for UV Shielding and Enhanced Yeast Surviva](#section4)
[Week 1: Melanin Biosynthesis and Biopolymer Fabrication](#section4-1)
[Week 2: UV-Shielding Evaluation and Yeast Survival Testing](#section4-2)
[Cloning and Preliminary Assessment of Rad51 Overexpression for Radiation Resistance](#section5)
[Week 1: Rad51 Cloning and PCR Optimization](#section5-1)
[Week 2: Preliminary Functional Assay and Future Planning](#section5-2)
[Construction of DSUP Expression Plasmid in Saccharomyces cerevisiae for Radiation Resistance Applications](#section6)
[Week 1](#section6-1)
[Week 2](#section6-2)

## Construction and Characterization of GAT1 Knockout in S. cerevisiae

### Week 1: Primer Design and Preparation; (05.04.2025 - 11.04.2025)

* Retrieved the GAT1 gene sequence (YFL021W) from the Saccharomyces Genome Database (SGD).
* Designed primers with ~50 bp homology arms flanking the GAT1 open reading frame.

Del\_GAT1\_fwd:
**ACATATATATAGGTGTGTGCCACTCCCGGCCCCGGTATTAGCATG**CAGCTGAAGCTTCGTACGC

Del\_GAT1\_rev:
**GCGGACATGGAAAGAAGCGAGTACTTTTTTTTTTTGGGGGATCTA**GCATAGGCCACTAGTGGATCTG

(homologous flanking sequences are in bold letters)

* Ordered custom primers from a commercial supplier for disruption cassette amplification.
* Prepared competent E. coli DH5α cells for plasmid propagation and storage.
* Prepared SC–URA, SC–LEU, and YNB + ammonium sulfate media for yeast culture and selection.
* Autoclaved agar separately from YNB/ammonium sulfate to prevent precipitation.
* Extracted genomic DNA from S. cerevisiae CEN.PK1D for use as a PCR verification template.

### Week 2: Disruption Cassette Construction & Yeast Transformation; (13.04.2025 - 18.04.2025)

* PCR-amplified loxP–URA3–loxP cassette from the pUG72 plasmid using the designed primers.
* Confirmed PCR amplification by agarose gel electrophoresis; observed a single, clear band of the expected size.
* Purified the PCR product using a gel extraction kit.
* Transformed S. cerevisiae CEN.PK1D with the linear loxP–URA3–loxP disruption cassette using the lithium acetate method.
* Plated transformed cells onto SC–URA plates for selection of positive transformants.
* Incubated plates at 30°C and monitored colony growth for 2–3 days.

### Week 3: Verification of GAT1 Knockout; (20.04.2025 - 26.04.2025)

* Picked well-isolated colonies from SC–URA selection plates.
* Performed colony PCR using flanking primers to verify successful integration at the GAT1 locus.
* Analyzed PCR band sizes via agarose gel electrophoresis to confirm correct GAT1::URA3 replacement.
* Cultured confirmed knockout strains in SC–URA broth for propagation.
* Prepared glycerol stocks of verified transformants for long-term storage at –80°C.
* Propagated pBF3036 (Cre recombinase plasmid) in E. coli, isolated plasmid DNA for subsequent yeast transformation.

### Week 4: URA3 Marker Excision; (28.04.2025 - 30.04.2025)

* Transformed GAT1::URA3 strains with pBF3036 (Cre recombinase plasmid).
* Selected transformants on SC–LEU plates to maintain plasmid selection.
* Induced Cre recombinase expression to excise the URA3 marker via loxP sites.
* Replica-plated transformants onto SC–URA plates to identify colonies that lost URA3.
* Colonies that failed to grow on SC–URA were identified as ΔGAT1 mutants.
* Validated URA3 excision and GAT1 deletion through PCR verification using external primers.

### Week 5: Phenotypic Testing – Nitrogen Analysis; (02.05.2025 - 10.05.2025)

* Cultured both wild-type (WT) and delGAT1 strains overnight in SC–URA medium.
* Subcultured cells to an initial OD₆₀₀ = 0.05 and incubated until mid-log phase.
* Harvested cells by centrifugation for Kjeldahl nitrogen analysis.
* Conducted digestion, neutralization, and distillation steps.
* Performed UV Spectroscopy to measure OD 420 of the digested mixture.
* These steps were performed to determine total nitrogen content.
* Converted nitrogen percentage to crude protein using the standard factor of 6.25.
* Compared the total nitrogen and protein content between WT and ΔGAT1 strains.
* Observed reduced nitrogen and protein content in ΔGAT1 mutants, confirming GAT1’s regulatory role in nitrogen metabolism.

## Enhancement of Nitrogen Assimilation in ΔGAT1 S. cerevisiae via GDH1 and GLN1 Overexpression

### Week 1: Target Selection and Experimental Design; (19.05.2025 - 24.05.2025)

* Reviewed background on nitrogen assimilation in S. cerevisiae: ~85 % of nitrogen for amino acid biosynthesis originates from the amino group of glutamate, and ~15 % from the amide group of glutamine.
* Selected GDH1 (Systematic Name: YOR375C, SGD ID: SGD:S000005902) (glutamate dehydrogenase) and GLN1 (Systematic Name: YPR035W, SGD ID: SGD:S000006239) (glutamine synthetase) as target genes for overexpression to improve nitrogen uptake and protein synthesis.
* Designed primers and collected gene sequences for cloning.

(Restriction sites are underlined)

Primers for GDH1;
Fwd\_GDH1\_BamHI: CGCGGATCCATGTCAGAGCCAGAATTTC
Rev\_GDH1\_Xhol: CCGCTCGAGTTAAAATACATCACCTTGGTC

Primers for GLN1;
Fwd GLN1\_BamHI: CGCGGATCCATGGCTGAAGCAAGCATC
Rev\_GLN1 Xhol: CCGCTCGAGTTATGAAGATTCTCTTTCAAATTCC

* Prepared competent S. cerevisiae ΔGAT1 (CEN.PK 1D) cells for transformation.
* Set up plasmid maps and expression constructs under suitable promoters.

### Week 2: Plasmid Construction and Yeast Transformation; (25.05.2025 - 30.05.2025)

* Constructed recombinant plasmids carrying the GDH1 and GLN1 genes.
* Verified constructs by restriction digestion and agarose gel electrophoresis.
* Transformed S. cerevisiae ΔGAT1 cells with GDH1 and GLN1 plasmids separately.
* Performed selection on the URA dropout minimal medium to isolate transformants.
* Confirmed positive colonies by colony PCR and stored glycerol stocks for both transformants and control (ΔGAT1).
* Began pre-cultures for growth and expression analysis.

### Week 3: Culture Inoculation and Expression Verification; (01.06.2025 - 07.06.2025)

* Inoculated triplicate cultures of delGAT1, delGAT1 + GDH1, and delGAT1 + GLN1 in synthetic URA dropout minimal medium for comparative growth studies.
* Monitored OD₆₀₀ values at regular intervals to track growth kinetics.
* Extracted total RNA and verified GDH1 and GLN1 expression by PCR amplification; observed clear bands on agarose gel, confirming successful overexpression.
* Prepared cultures for biochemical assays and stored aliquots for nitrogen estimation.

### Week 4: Nitrogen and Protein Analysis; (09.06.2025 - 16.06.2025)

* Harvested all three strains at mid-log phase for nitrogen and protein analysis.
* Performed the Kjeldahl method for total nitrogen determination:
  + Step 1 – Digestion with concentrated H₂SO₄ to convert organic N → NH₄⁺.
  + Step 2 – Neutralization with NaOH and distillation.
  + Step 3 – Performed UV Spectroscopy to measure OD 420 of the digested mixture.
* Calculated nitrogen (%) and converted to protein content using the factor 6.25.
* Recorded OD₆₀₀ and dry cell weight for biomass comparison.

## Engineering S. cerevisiae for Nitrogen Recycling and Preliminary Assessment of Enhanced Nitrogen Assimilation

### Week 1: Engineering Yeast for Nitrogen Recycling; (18.06.2025 - 24.06.2025)

* Modified S. cerevisiae strains were designed to recycle nitrogenous waste into usable protein forms, contributing to a circular bioeconomy framework. This approach has potential applications in addressing global protein deficiency and sustaining closed environments such as the International Space Station (ISS), where efficient waste utilization and nutrient recovery are vital for astronaut health and muscle mass maintenance.
* Recombinant plasmids containing the GDH1 and GLN1 genes were successfully constructed and introduced into both the wild-type S. cerevisiae and the ΔGAT1 mutant strain. Transformants were screened and confirmed using selective media, setting the foundation for comparing nitrogen metabolism in the presence and absence of GAT1, a key transcriptional regulator.

### Week 2: Preliminary Assessment of Nitrogen Assimilation; (28.06.2025 - 04.07.2025)

* Both modified and unmodified yeast strains were grown in urea-based minimal medium to assess their growth performance and nitrogen assimilation efficiency. Early results suggested promising outcomes, particularly in the GDH1+ΔGAT1 strain, which exhibited enhanced nitrogen utilization compared to the unmodified parent strain.
* However, the results remain preliminary, as time constraints prevented repeat trials. Further testing and data validation are planned post–wiki freeze to confirm the observed improvements and ensure experimental reliability.

## Development of Melanin–PVA Sheets for UV Shielding and Enhanced Yeast Survival

### Week 1: Melanin Biosynthesis and Biopolymer Fabrication; (03.08.2025 - 13.08.2025)

* While studying amino acid utilization in P. vulgaris, a serendipitous observation was made — cultures grown with L-tyrosine produced eumelanin through the dopamine (catecholamine) biosynthetic pathway. Recognizing its UV-shielding potential, this pathway was integrated into our radiation protection design.
* After evaluating several coating strategies, we finalized the idea of embedding eumelanin in a PVA matrix. PVA was selected for being lightweight, porous, and chemically inert to NaOH, aiding both payload reduction and thermal stability.
* During the week, P. vulgaris was cultured for seven days, progressively darkening to black, confirming melanin synthesis. The pigment was extracted, purified, and dried, then dissolved in NaOH and mixed with 10 w/v % PVA to yield 0.05 w/v % eumelanin–PVA solution. After sonication, the mixture was cast and dried into uniform biopolymer sheets for testing.

### Week 2: UV-Shielding Evaluation and Yeast Survival Testing; (15.08.2025 - 21.08.2025)

* Initial UV-shielding tests were performed using a transilluminator. The PVA + melanin sheet visibly blocked more UV light compared to the PVA + NaOH control, confirming its optical protective properties.
* For quantitative testing, S. cerevisiae spread plates (10⁴ dilution) were exposed to UV for 1 h inside a LAF hood:
  + PVA + melanin sheet: 1.85 × 10⁴ CFUs
  + PVA + NaOH control: negligible CFUs
    Uncovered control (outside LAF): 1.17 × 10⁴ CFUs
* The melanin-coated plate showed higher yeast survival and no visible damage, while the control sheet exhibited burning and edge deformation.
* Plans were made to collaborate with Dr. Joe Ninan (TIFR) for high-altitude balloon testing to simulate upper-atmosphere radiation.

## Cloning and Preliminary Assessment of Rad51 Overexpression for Radiation Resistance

### Week 1: Rad51 Cloning and PCR Optimization; (21.09.2025 - 28.09.2025)

* The Rad51 sequence was retrieved from the SGD database. A pRS426 high-copy plasmid was chosen for gene expression, along with a GPD constitutive promoter and a CYC1 terminator. The GPD promoter ensures continuous expression of Rad51, which is critical for radiation resistance in space, while the CYC1 terminator prevents downstream gene expression interference. GFP was included as a screening marker to confirm Rad51 expression.
* Primers were designed for PCR amplification of Rad51:

Forward Primer (Fwd\_RAD\_BamHI): GCGGGATCCATGTCTCAAGTTCAAGAACAAC
Reverse Primer (Rev\_RAD\_HindIII): GCGAAGCTTCTACTCGTCTTCTTCTCTGG

* Genomic DNA was extracted from wild-type yeast and quantified using a Nanodrop. Initial PCR attempts produced dimers and low yield despite repeated genomic DNA extractions and 9 PCR cycles. After further optimization, a successful amplification of Rad51 was obtained.

### Week 2: Preliminary Functional Assay and Future Planning; (29.09.2025 - 04.10.2025)

* A spread plate assay was set up to compare wild-type yeast and yeast with overexpressed Rad51 on YPD agar. While the test is ongoing in the wet lab, data collection and validation will continue after the wiki freeze.
* Lessons from this work suggest that future teams could achieve Rad51 overexpression more efficiently by:
  + Using a synthesized gene sequence instead of genomic amplification.
  + Modifying the native promoter in the genome to directly upregulate Rad51 expression.

## Construction of DSUP Expression Plasmid in Saccharomyces cerevisiae for Radiation Resistance Applications

### Week 1: Week 1 ; (21.09.2025 - 29.09.2025)

* Based on the literature and BBa\_K2195000 from the iGEM registry, DSUP has been reported to improve host organism survival under radiation, even in non-native systems. The DSUP sequence was codon-optimized for Saccharomyces cerevisiae and synthesized by Twist Biosciences. A GPD constitutive promoter was chosen to ensure continuous expression in space conditions, and a CYC1 terminator was included downstream.
* Clonal plasmids containing DSUP with XbaI and XhoI restriction sites were transformed into E. coli DH5α competent cells. After incubation, plasmid prep was performed. Simultaneously, a blank pRS426 plasmid was cultured in LB + Amp media and isolated after 16 hours at 37 °C. Both the DSUP-containing plasmids and the pRS426 plasmid were digested with XbaI and XhoI in Tango buffer for 4.5 hours, and the DSUP insert was separated using gel electrophoresis.
* The insert was purified and stored at –20 °C, while challenges with pRS426 digestion were addressed by switching to FD (Fast Digested) buffer after one week of iteration.

### Week 2; (30.09.2025 - 04.10.2025)

* Due to time constraints, only the ligation of the DSUP insert into the pRS426GPD backbone was completed by the wiki freeze. Transformation of this plasmid into S. cerevisiae will be performed post-wiki freeze.
* For future functional testing, a UV facility is available to evaluate growth under controlled radiation conditions, in compliance with iGEM containment rules.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering

#### At the Heart of every Innovation, Engineering at its Best

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Exploring the engineering backbone of our project. Every problem can be solved when one changes their perspective on the problem. Showcasing our technical rigor, creativity, real-world constraints, and various engineering approaches of our team, converging to shape our solution.

![page-centrepiece](https://static.igem.wiki/teams/5893/pagelogo/engineering.webp)

[Protein Production](#section1)
[DBTL-1](#section1-1)
[DBTL-2](#section1-2)
[References](#references1)
[Radiation Resistance](#section2)
[Rad51](#section2-1)
[DSUP](#section2-2)
[References](#references2)
[Melanin Shielding](#section3)
[Melanin Films](#section3-1)
[References](#references3)

## Protein Production

### DBTL-1

#### Design:

The GAT1 gene (Systematic Name: YFL021W, SGD ID: SGD: S000001873) was selected as a target. The goal of this engineering cycle is to demonstrate whether its deletion optimizes nitrogen metabolism to maximize amino acid and protein production from urea, supporting our goal of creating a sustainable single-cell protein source for astronauts. The GAT1 gene was deleted to simplify nitrogen catabolite repression control and evaluate the contribution of GAT1-dependent regulation in urea utilization.

In *S. cerevisiae*, nitrogen metabolism is regulated by Nitrogen Catabolite Repression (NCR), which enables preferential use of easily assimilable nitrogen sources like glutamine and ammonia. The GATA transcription factors Gln3p and Gat1p activate, while Dal80p and Nil2p repress NCR-sensitive genes. Under nitrogen-rich conditions, Ure2p sequesters Gln3p in the cytoplasm, but during nitrogen scarcity, it relocates to the nucleus to activate key assimilation genes such as GDH2 and GLN1. The TOR pathway modulates this process by controlling Gln3p and Gat1p localization in response to nitrogen availability.

Two enzymes, glutamate dehydrogenase (Gdh1p) and glutamine synthetase (Gln1p), play central roles in assimilating ammonium into glutamate and glutamine. Their activities rise under nitrogen-limited conditions and are inhibited by intracellular glutamine when nitrogen is abundant, maintaining metabolic balance. Thus, it was hypothesised that if the genes controlling these enzymes are overexpressed, in addition to the deletion of the GAT1 gene, the intracellular protein content would increase without having limit control.[1]

![nitrogen-pathway](https://static.igem.wiki/teams/5893/engineering/1-nitrogen.webp)

#### Build:

The GAT1 gene (Systematic Name: YFL021W, SGD ID: SGD: S000001873) [10] in *S. cerevisiae* (strain CENPK1D) was disrupted using a homology-based gene knockout strategy to enable precise replacement of the target locus. This approach employed the URA3 selection marker derived from the pUG72 plasmid, flanked by loxP recombination sites. To generate the disruption cassette, the loxP–URA3–loxP sequence was PCR-amplified using primers containing approximately 50 base pairs of homology to the genomic regions immediately upstream and downstream of the GAT1 open reading frame.

Primers used for amplification:

Del\_GAT1\_fwd: **ACATATATATAGGTGTGTGCCACTCCCGGCCCCGGTATTAGCATG**CAGCTGAAGCTTCGTACGC

Del\_GAT1\_rev: **GCGGACATGGAAAGAAGCGAGTACTTTTTTTTTTTGGGGGATCTA**GCATAGGCCACTAGTGGATCTG

(homologous flanking sequences are in bold letters)

These homology arms facilitated targeted integration of the cassette into the yeast genome via homologous recombination, effectively replacing the native GAT1 gene with the URA3 marker.

![nitrogen-pathway](https://static.igem.wiki/teams/5893/engineering/new-2-rxn-1.webp)

The linear PCR product was introduced into yeast cells using the lithium acetate transformation method. Successful transformants were selected on synthetic complete (SC) media lacking uracil (SC–Ura), confirming URA3 integration at the GAT1 locus. This strategy enabled efficient, targeted gene disruption, making it well-suited for downstream functional genomic studies. To excise the URA3 selection marker following its integration at the GAT1 locus, the knockout strain was co-transformed with plasmid pBF3036, which carries an inducible Cre recombinase gene. Cre recombinase catalyzes site-specific recombination between the loxP sites flanking the URA3 cassette, leading to its excision from the genome.

![nitrogen-pathway](https://static.igem.wiki/teams/5893/engineering/new-3-rxn-2.webp)

![nitrogen-pathway](https://static.igem.wiki/teams/5893/final/final/1.webp)

Transformants were selected on synthetic complete (SC) media supplemented with leucine to maintain the Cre-expressing plasmid. Induction of Cre expression was followed by replica plating of the colonies onto SC–Ura medium to screen for URA3 loss. Colonies that failed to grow on SC–Ura were indicative of successful marker excision. These colonies were considered confirmed GAT1 deletion mutants, in which the target gene had been disrupted and the URA3 marker subsequently removed.

![nitrogen-pathway](https://static.igem.wiki/teams/5893/final/final/2.webp)

#### Test:

*S. cerevisiae* strain CEN.PK1D (Parent) and del GAT1 strain were initially cultured overnight in yeast minimal media [synthetic complete medium lacking uracil (SC–Ura)].

![nitrogen-pathway](https://static.igem.wiki/teams/5893/final/final/3.webp)

The overnight cultures were subcultured, and the optical density at 600 nm (OD₆₀₀) was measured. This OD was correlated to the dry cell weight using experimentally observed correlations. Cell pellets from both the mutant and control strains were harvested and analyzed for total nitrogen content using Kjeldahl digestion followed by the micro-Kjeldahl method. The measured nitrogen percentage was then converted to crude protein content using a conversion factor of 6.25.

![nitrogen-pathway](https://static.igem.wiki/teams/5893/final/final/4.webp)

#### Learn:

Overall deletion of GAT1 caused an increase in the protein content as hypothesised.

### DBTL-2

##### Iteration 1

#### Design:

In *S. cerevisiae*, the majority of nitrogen used for amino acid biosynthesis is derived from glutamate and glutamine. Approximately 85% of the cellular nitrogen originates from the amino group of glutamate, while the remaining 15% is contributed by the amide group of glutamine. These two amino acids serve as central nitrogen donors, playing a crucial role in maintaining nitrogen balance and supporting protein synthesis within the cell. In the present study, we sought to increase nitrogen content in the Del GAT1 + S. cerevisiae CENPK.1D by overexpressing enzymes involved in nitrogen metabolism. Through strategic overexpression of key nitrogen metabolism genes GDH1 and GLN1, the modified strains are expected to exhibit improved growth and biomass production, thereby boosting the potential of these yeasts as Single Cell Protein (SCP) sources.[1]

#### Build:

Recombinant plasmids containing the GDH1 (Systematic Name: YOR375C, SGD ID: SGD:S000005902) [BBa\_25TI69KR](https://registry.igem.org/parts/bba-25ti69kr)[10] and GLN1 (Systematic Name: YPR035W, SGD ID: SGD:S000006239) [BBa\_25GT9CLB](https://registry.igem.org/parts/bba-25gt9clb) [11] genes were initially isolated from S. cerevisiae CEN.PK1D genomic DNA using the following primers.

Primers for GDH1:

Fwd\_GDH1\_BamHI: CGC**GGATCC**ATGTCAGAGCCAGAATTTC

Rev\_GDH1\_Xhol: CCG**CTCGAG**TTAAAATACATCACCTTGGTC

Primers for GLN1:

Fwd\_GDH1\_BamHI: CGC**GGATCC**ATGGCTGAAGCAAGCATC

Rev\_GDH1\_Xhol: CCG**CTCGAG**TTATGAAGATTCTCTTTCAAATTCC

PCR conditions for amplification included an annealing temperature of 57°C and an extension time of 2 minutes, optimized for the sizes of GDH1 (1362 bp) and GLN1 (1110 bp), respectively.

![table 2.1.1](https://static.igem.wiki/teams/5893/final/final/5.webp)

![table 2.1.1](https://static.igem.wiki/teams/5893/final/final/6.webp)

These isolated genes were separately recombined with pRS426-GPD with GPD promoter and CYC1 terminator using the plasmid backbone digested with restriction enzymes BamHI and Xhol.

![table 2.1.1](https://static.igem.wiki/teams/5893/final/final/7.webp)

Recombinant plasmids containing the GDH1 and GLN1 genes were successfully introduced into the delGAT1 mutant. Following the transformation, positive clones were identified through selection on appropriate media (minimal media).

![table 2.1.1](https://static.igem.wiki/teams/5893/final/final/8.webp)

This experimental setup allows a direct comparison of GDH1 and GLN1 activity in the presence of GAT1, a central transcriptional regulator involved in nitrogen metabolism, in comparison to the original *S. cerevisiae strain*.

#### Test:

By examining these strains side-by-side, we can better understand how GAT1 influences the cellular response to enhanced nitrogen assimilation. Targeted manipulation of key metabolic genes can significantly improve protein production, even in strains lacking native regulatory elements. To evaluate the impact of GDH1 and GLN1 overexpression on protein accumulation in del GAT1 strain  *S. cerevisiae*, total nitrogen content was determined using the Kjeldahl method. The delGAT1 mutant strains were transformed with either GDH1, GLN1 and grown in triplicate in synthetic URA dropout minimal medium. Cultures were harvested at mid-log phase, and nitrogen content was converted to protein using a conversion factor of 6.25.

![proteinchart3](https://static.igem.wiki/teams/5893/final/final/9.webp)

![proteinchart3](https://static.igem.wiki/teams/5893/final/final/10.webp)

#### Learn:

Overexpression of GDH1 with the deleted GAT1 gene gave the most protein content, followed by the overexpressed GLN1 with the deleted GAT1 gene and the deleted GAT1 strain. This further reinforces our initial hypothesis.

##### Iteration 2

#### Design:

These results indicate that the modified *S. cerevisiae* strains have the potential to be utilized for nitrogenous waste recycling. Implementing this circular economy approach could contribute to addressing global protein deficiency challenges. Moreover, this principle holds particular promise for closed environments such as the International Space Station (ISS), where efficient resource recovery and waste utilization are of critical importance while also addressing the pressing issue of protein deficiency in astronauts, leading to a reduction in muscle mass.

#### Build:

Recombinant plasmids containing the GDH1 and GLN1 genes were successfully introduced into both the wild-type *S. cerevisiae* strain and the delGAT1 mutant. Following the transformation, positive clones were identified through selection on appropriate media. This experimental setup allows a direct comparison of GDH1 and GLN1 activity in the presence of GAT1, a central transcriptional regulator involved in nitrogen metabolism, in comparison to the original S. cerevisiae strain.

#### Test:

The same tests were carried out for the modified and unmodified strains, but in a urea medium.

![ureaproteinchart](https://static.igem.wiki/teams/5893/final/final/11.avif)

#### Learn:

This shows us promising results, especially in the GDH1+delGAT1 strain when compared to the unmodified parent strain

There is a high possibility that the results of this experiment are erroneous; there was no opportunity to reperform this due to the time constraint. Further testing will be carried out post-wiki-freeze.

Show References

1. Magasanik, B., & Kaiser, C. A. (2002). Nitrogen regulation in *Saccharomyces cerevisiae*.
   *Gene*, **290(1–2)**, 1–18.
   [Link](https://doi.org/10.1016/S0378-1119%2802%2900558-9)
2. Georis, I., Feller, A., Vierendeels, F., & Dubois, E. (2009).
   The yeast GATA factor Gat1 occupies a central position in nitrogen catabolite repression-sensitive gene activation.
   *Molecular Microbiology*, **73(5)**, 1002–1017.
   [Link](https://doi.org/10.1111/j.1365-2958.2009.06826.x)
3. Kennedy, S. R., Loeb, L. A., & Herr, A. J. (2012).
   Somatic mutations in aging, cancer and neurodegeneration.
   *Mechanisms of Ageing and Development*, **133(4)**, 118–126.
   [Link](https://doi.org/10.1016/j.mad.2012.01.005)
4. Alexandrov, L. B., Kim, J., Haradhvala, N. J., Huang, M. N., Ng, A. W. T., Boot, A., et al.; PCAWG Mutational Signatures Working Group; PCAWG Consortium. (2020).
   The repertoire of mutational signatures in human cancer.
   *Journal of Biological Chemistry*, **295(14)**, 476–489.
   [Link](https://doi.org/10.1016/j.jbc.2019.12.014)
5. Entian, K.-D., & Kötter, P. (2007).
   Yeast genetic strain and plasmid collections.
   In *Methods in Microbiology* (Vol. 36, pp. 629–666). Academic Press.
   [Link](https://doi.org/10.1016/S0580-9517%2806%2936025-4)
6. Mumberg, D., Müller, R., & Funk, M. (1995).
   Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.
   *Gene*, **156(1)**, 119–122.
   [Link](https://doi.org/10.1016/0378-1119%2895%2900037-7)
7. Gueldener, U., Heinisch, J., Koehler, G. J., Voss, D., & Hegemann, J. H. (2002).
   A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.
   *Nucleic Acids Research*, **30(6)**, e23.
   [Link](https://doi.org/10.1093/nar/30.6.e23)
8. Fang, F., Salmon, K., Shen, M. W., Aeling, K. A., Ito, E., Irwin, B., Tran, U. P., Hatfield, G. W., Da Silva, N. A., & Sandmeyer, S. (2011).
   A vector set for systematic metabolic engineering in *Saccharomyces cerevisiae*.
   *Yeast*, **28(2)**, 123–136.
   [Link](https://doi.org/10.1002/yea.1824)
9. Saccharomyces Genome Database. GAT1.
   [Link](https://www.yeastgenome.org/locus/Gat1)
10. Saccharomyces Genome Database. GDH1.
    [Link](https://www.yeastgenome.org/locus/S000005902)
11. Saccharomyces Genome Database. GLN1.
    [Link](https://www.yeastgenome.org/locus/S000006239)

## Radiation Resistance

### Rad51

#### Design:

We identified the sequence of RAD51 from the SGD. And planned to use pRS426 high copy plasmid along with GPD promoter and a CYC1 terminator. The GPD is a highly used strong constitutive promoter which will drive continuous expression of our target RAD51 gene and will not be dependent on external stimuli to regulate its expression. The logic behind using a constitutive promoter is that in space, the organism is always exposed to some type of harmful radiation so it will require continuous expression of the gene. While the CYC1 terminator will stop the expression of genes to the downstream of our target and GFP. The GFP will be used as a screening marker to confirm the expression of RAD51.(fig. 1.2)

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/1.avif)

#### Build:

The primers for RAD51 were designed for PCR amplification of the gene.

**Forward Primer**- Fwd\_RAD\_BamHI: GCGGGATCCATGTCTCAAGTTCAAGAACAAC

**Reverse Primer**- Rev\_RAD\_HindIII: GCGAAGCTTCTACTCGTCTTCTTCTCTGG

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/2.avif)

The genomic DNA of a wild type yeast was extracted for PCR amplification of RAD51. After the genomic dna extraction the concentration was confirmed by Nanodrop machine. (fig 1.3)

The initial iteration of PCR was successful but the yield was too low. So we adjusted the PCR conditions but it did not yield any results. The team performed 9 PCR cycles as well as 3 genomic DNA extractions.The PCR continuously yielded Dimers in amplification but after a few iterations, we got successful iteration.
The Restriction Digestion and further steps of this cycle are ongoing but would not be complete until wiki freeze.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/3.avif)

#### Test:

The modified strain was to be tested by performing a spread plate on a YPD+agar media plate, one streaked with the wild type yeast and one with overexpressed RAD51. It is being continued in the wet lab but the build would not be complete until wiki freeze.

#### Learn:

Future teams can try to overexpress RAD51 by various other means such as by using synthesized sequence of the gene or by directly changing the promoter in genomic DNA to upregulate its expression as a faster way to overexpress it.

### DSUP

#### Design:

According to the literature survey and Part: BBa\_K2195000 from iGEM registry, along with other references [1][2] we can confirm that DSUP has the ability to affect the survival rate in host organisms even while not being native to the organism. Hence we did the Codon optimization of the sequence of DSUP gene for *Saccharomyces cerevisiae*. This was then synthesized by Twist Biosciences.

We planned to use a GPD constitutive promoter since the logic behind using a constitutive promoter is that in space, the organism is always exposed to some type of harmful radiation so it will require continuous expression of the gene. DSUP is used along with CYC1 terminator.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/4.avif)

#### Build:

The clonal plasmids had restriction sites Xba1 and Xho1. These clonal plasmids were transformed into *E.coli* DH5α competent cells. After incubating this transformed strain, plasmid prep was done for this Dh5α strain.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/5.avif)

Simultaneously, a blank Dh5α containing pRS426 plasmid was inoculated in 7ml of of LB+Amp media.

After 16hours of incubation at 37 degree celsius, plasmid prep was done and the pRS426 plasmid was isolated.

The clonal plasmids containing DSUP insert along with pRS426 plasmid were digested along with Tango buffer and Restriction enzymes Xba1 and Xho1 for 4.5hrs. After digestion, the insert from clonal plasmid containing DSUP was separated using Gel Electrophoresis.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/6.avif)

The insert was then purified and stored at -20 degrees celsius.

Until the wiki freeze we could only do the ligation. Transformation of this plasmid vector containing pRS426GPD backbone and DSUP gene insert into Saccharomyces cerevisiae will be done post wiki freeze. And the work will continue on this DBTL cycle even post wiki freeze.

#### Test:

Since there are strict rules from iGEM of containing strains within the lab even for testing, we have a UV facility in the lab to test its growth under radiation setup.

#### Learn:

The learnings of this part will help us understand if the hypothesis stands true for DSUP being expressed in *Saccharomyces cerevisiae* as well. If the system works, future teams working on Space village projects can take advantage of this system to use the modified *Saccharomyces cerevisiae* as a chassis for biomanufacturing in space.

Show References

1. [0]Singh NP, Dhawan J, Chadha S. Understanding the unusual radiation resistance of insect cells: Valuable lessons for stress biology and oncology studies.
   *Annals of Biological Research*. 2019;10(1):1-5.
2. [1]Anoud M, Delagoutte E, Helleu Q, Couté Y, Guidetti R, Rebecchi L, Giovannangeli C, De Cian A, Concordet JP.
   Comparative transcriptomics reveal a novel tardigrade-specific DNA binding protein induced in response to ionizing radiation.
   *eLife*. 2024 Jul 9;13:RP92621.
   doi: [10.7554/eLife.92621](https://doi.org/10.7554/eLife.92621)
3. [2]Chavez C, Cruz-Becerra G, Fei J, Kassavetis GA, Kadonaga JT.
   The tardigrade damage suppressor protein binds to nucleosomes and protects chromatin from hydroxyl radicals.
   *eLife*. 2019 Sep 30;8:e47682.
   doi: [10.7554/eLife.47682](https://doi.org/10.7554/eLife.47682)

## Melanin Shielding

### Melanin Film

#### Design:

A serendipitous discovery made in our lab of melanin production was made while studying the nutritional requirements of different amino acids of *P. vulgaris*. When *P. vulgaris* was cultured in a medium in the presence of L-tyrosine, it gave eumelanin via the dopamine (catecholamine) biosynthetic pathway

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/1m.avif)

To capitalise on this finding, we integrated this eumelanin production pathway into our design for radiation protection, leveraging the well-established UV-shielding properties of eumelanin to enhance the resilience of our chassis against space radiation. We decided to coat our bioreactor with a layer of eumelanin for additional radiation resistance. We ideated a system in which a eumelanin-suspended liquid could be circulated on the outside of the bioreactor, but this was soon discarded as setting up such a mechanism for eumelanin circulation would need to be extremely sophisticated and would not be payload-effective. Another method of dispersing the eumelanin in PMMA, painting it onto the bioreactor and allowing it to dry was hypothesised. This was discarded as we were unsure of getting an even coat of paint onto the bioreactor.

Finally, we arrived at the idea of dispersing melanin into a PVA sheet as:

1. PVA is well known to be lightweight and porous which would help us with payload reduction and heat dissipation(from the reactor)
2. PVA was chosen as it is inert to the NaOH needed to dissolve the eumelanin
3. A reusable PVA sheet seemed to be the most sustainable method for radiation protection in outer space settings

These sheets would be synthesized on earth and consequently be sent as replacements for the bioreactor in space when required.

#### Build:

Melanin was produced using the above-mentioned metabolic pathway present in *P. vulgaris*. *P. vulgaris* was cultured in a medium containing L-tyrosine and incubated for a week. The culture flask gradually turned darker until it became almost black, indicating the presence of eumelanin. This eumelanin was then extracted from the culture medium as documented in the protocols

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/2m.avif)

The dried eumelanin powder was consequently used for making a biopolymer with PVA. The eumelanin was dissolved in a NaOH solution and added to the PVA solution(detailed in the lab notebook). This solution was then dried to make the biopolymer sheets of 10w/v% PVA and 0.05w/v% eumelanin. Further testing was done on the eumelanin itself, as well as the biopolymer sheet was carried out as detailed below.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/3m.avif)

#### Test:

A preliminary visual test was done to evaluate the effectiveness of this eumelanin-containing biopolymer in UV shielding by placing it on a transilluminator and comparing it with a control sheet.

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/4m.avif)

This shows us that our biopolymer sheet is very effective at reflecting and absorbing UV, visible light.
UV shielding ability quantification of the sheets
spread plates of *S. cerevisiae* were made of dilution 10^4, 300 microlitres of diluted culture was plated onto an agar medium, exposed to UV light in a LAF for 1 hour.
The first plate was covered with a sheet of PVA+melanin+NaOH
The second plate was covered with a control sheet of PVA+NaOH
Both of these were placed in the LAF for testing
The third uncovered plate was placed outside the LAF as a control
Subsequently, all three spread plates were incubated at 37degC for 2 days

Results obtained:

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/5m.avif)

* Control Plate (uncovered, outside LAF): Showed normal growth, as it was not exposed to UV radiation. (1.17 x 104 CFUs)
* Control Plate (PVA + NaOH only): Showed reduced colony growth, indicating that PVA alone provides minimal UV shielding.(Negligible number of CFUs)
* Test Plate (PVA + melanin + NaOH): Displayed significantly higher colony survival under UV compared to the PVA-only sheet. Interestingly, the melanin-covered plate showed even more growth than the plate outside UV, suggesting that brief, shielded UV exposure enhanced yeast survival.(1.85 x 104 CFUs)

This observation is supported by previous findings that *S. cerevisiae* cells pre-exposed to low UV doses develop an epigenetically inherited resistance, resulting in enhanced survival upon subsequent UV exposure [2].

An important observation was made- after an hour of UV exposure, the control sheet showed visible burns and crumpling at the edges, while the PVA+melanin sheet showed no such damage

![ureaproteinchart](https://static.igem.wiki/teams/5893/engineering/final2/6m.avif)

Our research will involve a collaboration with Dr. Jo Ninan from the Tata Institute of Fundamental Research (TIFR) to further test the efficiency of our newly synthesized PVA + melanin sheets. Due to time constraints, this phase of the experiment will be conducted at a later date.

In this planned experiment, we will utilize Dr. Ninan's expertise in balloon testing to evaluate our material's protective properties. Plates of *S. cerevisiae* ( baker’s yeast, not our lab strains) will be covered with the synthesized PVA + melanin sheets. These covered plates will then be attached to a high-altitude balloon and sent into the upper atmosphere.

The primary objective of this experiment is to assess the viability of the yeast on the plates after being exposed to the extreme environmental conditions of the upper atmosphere, such as low pressure, cold temperatures, and increased cosmic and ultraviolet radiation. We hypothesize that the melanin component in the sheets will provide significant protection, thereby preserving the viability of the *S. cerevisiae*. The results of this test will provide crucial data on the durability and protective efficiency of our novel material.

#### Learn

* An issue of uneven melanin distribution in the sheets was observed in the initial stages; for this reason, we sonicated the PVA+melanin solution before casting it for drying to ensure even distribution
* The higher CFU count in the irradiated sample shielded by the biopolymer might suggest some amount of encouragement in the growth of *S. cerevisiae* under a small amount of radiation
* The visible damage to the control sheet(caused by overheating due to UV radiation) in comparison to none in the sheet containing eumelanin in addition to the transilluminator, results clearly indicate a significant reflectance and absorption of UV radiation in these biopolymer sheets

Show References

1. iGEM Parts Registry. BBa\_K3633008. iGEM; [cited 2025/09/9].
   Available from:
   [https://parts.igem.org/Part:BBa\_K3633008](https://parts.igem.org/Part%3ABBa_K3633008)
2. Reardon, J., Hopkins, J., Sabatinos, S., & Dickinson, W.J. (2022).
   An epigenetically inherited UV hyper-resistance phenotype in *Saccharomyces cerevisiae*.
   *Epigenetics & Chromatin*, 15(1), 31.
   <https://doi.org/10.1186/s13072-022-00464-5>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

# Experiments

#### Testing Ideas, Validating Innovation

---

Diving into the experimental background of our project. Arising from a sense of curiosity, every experiment performed, every hypothesis has taken us a step closer towards bringing our idea to fruition. From growing bacterial cultures to editing their genes, iterative trouble shooting and data-driven insights.

![page-centrepiece](https://static.igem.wiki/teams/5893/pagelogo/experiment.webp)

[E. coli Protocols](#ecoli)
[Yeast Protocols](#yeast)
[Melanin Protocols](#melanin)
[DSUP Protocols](#dsup)
[General Protocols](#general)
[Nitrogen Analysis Protocols](#nitrogen)

## E. coli Protocols

[E. coli culture](https://static.igem.wiki/teams/5893/experiments/culturingecoli.pdf)
[Plasmid prep](https://static.igem.wiki/teams/5893/experiments/plasmidprepfromecoli.pdf)
[Competent Cells](https://static.igem.wiki/teams/5893/experiments/competentcell.pdf)

## Yeast Protocols

[YPD yeast](https://static.igem.wiki/teams/5893/experiments/culturingsaccharomyces-cerevisiae-ypd.pdf)
 [Minimal media](https://static.igem.wiki/teams/5893/experiments/culturing-saccharomyces-cerevisiae-minimal-media.pdf)
[S. cerevisiae genomic DNA extraction](https://static.igem.wiki/teams/5893/experiments/dna-isolation.pdf)

## Melanin Protocols

[Melanin PVA sheets](https://static.igem.wiki/teams/5893/experiments/melaninpvasheetmaking.pdf)
[Melanin purification](https://static.igem.wiki/teams/5893/experiments/melaninproductionandpurification.pdf)

## DSUP Protocols

[DSUP1](https://static.igem.wiki/teams/5893/experiments/dsuptransformationpart-1.pdf)
[DSUP2](https://static.igem.wiki/teams/5893/experiments/dsuptransformationpart2.pdf)

## General Protocols

[Gel electrophoresis](https://static.igem.wiki/teams/5893/experiments/gelelectrphoresis.pdf)
 [Restriction Digestion](https://static.igem.wiki/teams/5893/experiments/digestion.pdf)
[Gel purification](https://static.igem.wiki/teams/5893/experiments/gelpurification.pdf)
[Ligation](https://static.igem.wiki/teams/5893/experiments/ligation.pdf)
[PCR](https://static.igem.wiki/teams/5893/experiments/pcr.pdf)
[Colony PCR](https://static.igem.wiki/teams/5893/experiments/colonypcr.pdf)

## Nitrogen Analysis Protocols

[Kjeldahl](https://static.igem.wiki/teams/5893/experiments/kjeldahl.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

# Results

#### Our Journey, Quantified

---

The critical culmination of our project's journey. Presenting the data collected, the observations made, and the conclusions drawn from the experiments that we conducted in our pursuit for innovation. Whether confirming our hypothesis or challenging them, these results form the backbone of our scientific narrative.

![page-centrepiece](https://static.igem.wiki/teams/5893/pagelogo/results.webp)

[Nitrogen](#section1)
[DBTL - 1](#section1-1)
[DBTL 2 -Iteration 1-GLN1 GDH1 addition in Yeast minimal medium](#section1-2)
[DBTL 2- Iteration 2 - GLN1 GDH1 addition in Urea Medium](#section1-3)
[RAD51](#section2)
[DSUP](#section3)
[Melanin](#section4)

## Nitrogen

### DBTL - 1

#### Overview

The GAT1 gene (Systematic Name: YFL021W, SGD ID: SGD: S000001873) in *Saccharomyces cerevisiae* was selected as a target. We did this because it encodes Gat1p, a GATA-type transcription factor that plays a central role in regulating the metabolism of non-preferred nitrogen sources such as urea, proline, and allantoin. Unlike Gln3p, which broadly controls nitrogen catabolite repression (NCR), Gat1p provides more selective regulation, making it an ideal candidate for a controlled intervention in nitrogen metabolism. By targeting GAT1, we aimed to relieve repression on key nitrogen assimilation genes, including GDH2 and GLN1, thereby enhancing ammonium assimilation and intracellular nitrogen levels.

We deleted the gene using a homology-based knockout approach to enable precise replacement of the target locus.

A loxP–URA3–loxP cassette from plasmid pUG72 was integrated into the GAT1 locus via homologous recombination, followed by Cre–loxP-mediated marker excision using plasmid pBF3036.

The delGAT1 mutant and wild-type strains were then cultured in synthetic complete (SC–Ura) medium, and total nitrogen and crude protein contents were quantified using the Kjeldahl method to assess changes in intracellular protein levels.

## Hypothesis

It was hypothesized that the deletion of GAT1 gene which is a key activator in the Nitrogen Catabolite repressor system, would:

* Reduce the activation of genes responsible for the metabolism of urea sources not preferred by the cell which generally include Urea, Proline and Allantoin out of which our major target is Urea
* Redirects nitrogen flux away from urea breakdown and toward nitrogen incorporation
* reduces the cell's tendency to divert nitrogen into catabolic pathways under nitrogen stress and forces reliance on more efficient ammonium assimilation systems incorporating urea.

## Results

Optical density (OD₆₀₀) was measured and correlated to dry cell weight to standardize biomass before analysis.

The crude protein content was measured using harvesting and analysing cell pellets from both the mutant and control strains.

The method used was Kjeldahl digestion followed by the micro-Kjeldahl method. The measured nitrogen percentage was then converted to crude protein content using a conversion factor of 6.25.

It was observed that the mutant strains obtained had (12.38%) increase in protein content.

![Teamwork](https://static.igem.wiki/teams/5893/result/15.webp)

## Learnings

The results confirmed that GAT1 deletion positively impacted intracellular protein accumulation proving the initial hypothesis.

This suggests that disrupting Gat1p-mediated transcriptional activation in the NCR network can deregulate nitrogen assimilation, leading to enhanced utilization of non-preferred nitrogen sources.

### DBTL 2 - Iteration 1 GLN1 GDH1 addition in Yeast minimal medium

#### Overview

In *Saccharomyces cerevisiae*, the majority of nitrogen used for amino acid biosynthesis is derived from glutamate and glutamine. Approximately 85% of the cellular nitrogen originates from the amino group of glutamate, while the remaining 15% is contributed by the amide group of glutamine.

In the present study, we sought to increase nitrogen content in the **Del GAT1 + *S. cerevisiae* (CENPK.1D)** strain by overexpressing enzymes involved in nitrogen metabolism through strategic overexpression of key nitrogen metabolism genes *GDH1* and *GLN1*.

Recombinant plasmids containing GDH1 and GLN1 were introduced into the **del GAT1** mutant, and positive clones were identified through selection on appropriate media.

The media used was **URA Dropout Minimal Medium**, prepared with Yeast Nitrogen Base (0.67 g, without amino acids and nitrogen source), 2% dextrose (2 g), ammonium sulfate (0.5 g) as the nitrogen source, and sterile distilled water up to 100 mL. This medium was used for maintaining URA3-based transformants under nutrient-limited conditions.

#### Hypothesis

It was hypothesized that overexpressing GDH1 and GLN1 in the **del GAT1** mutant would further enhance nitrogen assimilation and intracellular protein accumulation.

GDH1 encodes NADPH-dependent glutamate dehydrogenase, which converts α-ketoglutarate and ammonium into glutamate.

GLN1 encodes glutamine synthetase, which synthesizes glutamine from glutamate and ammonium.

By increasing the activity of these key enzymes, we expected to boost the flow of nitrogen into amino acids and proteins, surpassing the protein accumulation observed in the **del GAT1** strain alone.

#### Results

The **del GAT1** mutant strains were transformed with either GDH1 or GLN1 and grown in triplicate in synthetic URA dropout minimal medium. Cultures were harvested at mid-log phase and analyzed.

To evaluate the impact of GDH1 and GLN1 overexpression on protein accumulation in *S. cerevisiae*, total nitrogen content was determined using the Kjeldahl method, and nitrogen content was converted to protein using a conversion factor of **6.25**.

It was observed that:

1. The **Del GAT1** only strain had a **14.83%** increase in protein content compared to the parent strain.
2. The **Del GAT1 + GLN1** strain had a **41.4%** increase in protein content compared to the parent strain.
3. The **Del GAT1 + GDH1** strain had a **262.65%** increase in protein content compared to the parent strain.

![Teamwork](https://static.igem.wiki/teams/5893/result/16.webp)

#### Learnings

The results confirmed that GAT1 deletion positively impacted intracellular protein accumulation proving the initial hypothesis

This suggests that disrupting Gat1p-mediated transcriptional activation in the NCR network can deregulate nitrogen assimilation, leading to enhanced utilization of non-preferred nitrogen sources

### DBTL 2- Iteration 2 - GLN1 GDH1 addition in Urea Medium

#### Overview

In this study, we sought to increase nitrogen content in the Del GAT1 + *S.cerevisiae* (CENPK.1D) by overexpressing enzymes involved in nitrogen metabolism through strategic overexpression of key nitrogen metabolism genes GDH1 and GLN1 in a different medium which is the Urea Medium which is prepared with Yeast Nitrogen Base (0.67 g, without amino acids and nitrogen source), 2% dextrose (2 g), urea (0.5 g) as the nitrogen source, and sterile distilled water up to 100 mL, used to assess nitrogen assimilation and recycling capability from urea.

#### Hypothesis

It was hypothesized that the del GAT1 + GDH1 and Del GAT1 + GLN1 strains would show enhanced utilization of urea as a nitrogen source compared to the Del GAT1 and the parent strain. The deletion of GAT1 was expected to relieve repression on non-preferred nitrogen assimilation genes, while GDH1 and GLN1 overexpression would increase flux through glutamate and glutamine pathways, boosting intracellular nitrogen content and protein accumulation even under suboptimal nitrogen conditions.

#### Results

All four strains were cultured in urea medium, and total nitrogen and crude protein content were determined using Kjeldahl digestion followed by the micro-Kjeldahl method, with protein calculated using a conversion factor of 6.25

It was observed that:

1. The Del GAT1 only strain had (9.67%) increase in protein content observed as compared to the parent strain
2. The Del GAT1 + GLN1 strain had (20.63%) increase in protein content observed as compared to the parent strain
3. The Del GAT1 + GDH1 strain had (23.53%) increase in protein content observed as compared to the parent strain

![Teamwork](https://static.igem.wiki/teams/5893/result/17.webp)

#### Learnings

The results support the potential of engineered strains for nitrogenous waste recycling, with the Del GAT1 + GDH1 strain showing the most promise.

However, due to time constraints, these experiments could not be repeated, and there is a possibility that the results may be inaccurate or require validation. Further testing and replication are planned post-wiki-freeze to confirm these trends. These preliminary findings highlight the feasibility of metabolic engineering strategies for improving SCP production from alternative nitrogen sources such as urea, which could be particularly valuable in resource-limited or closed environments like the ISS.

## RAD51

#### Overview

The radiation resistance of microorganisms offers key advantages for space cultivation systems. During an Integrated Human Practices (IHP) discussion with Dr. Suresh Naik (ISRO), the importance of addressing Galactic Cosmic Radiation (GCRs) in *S. cerevisiae* was highlighted. Literature review by the team concluded Tardigrades as the prime organisms for radiation survival due to their unique molecular adaptations.

One of them is the upregulation of DNA repair genes such as RAD51, a conserved recombinase involved in homologous recombination (HR), one of the two major DNA double strand break pathways. Since RAD51 is native to yeast, We started with that first…..We aimed to enhance DNA repair efficiency by overexpressing RAD51 under a constitutive GPD promoter in the pRS426 high-copy plasmid, with CYC1 terminator and GFP as a reporter.

#### Hypothesis

We hypothesized that overexpression of RAD51 in *S. cerevisiae* under the control of a constitutive promoter would enhance homologous recombination-based DNA repair, thereby improving radiation resistance. Thus, yeast cells carrying this construct should demonstrate greater survivability and genomic stability compared to wild-type strains when exposed to simulated radiation stress.

#### Results

Primers were designed for PCR amplification of the RAD51 gene using genomic DNA extracted from wild-type S. cerevisiae.

* Forward Primer (Fwd\_RAD\_BamHI): GCGGGATCCATGTCTCAAGTTCAAGAACAAC
* Reverse Primer (Rev\_RAD\_HindIII): GCGAAGCTTCTACTCGTCTTCTTCTCTGG

The modified strain test was planned by performing a spread plate on a YPD media agar plate, one of the wild yeast and one with overexpressed RAD51. A Nanodrop spectrophotometer was used to confirm the concentration of the extracted genomic DNA.

Multiple rounds—9 PCRs and 3 genomic DNA extractions—were performed but consistently resulted in primer dimer formation. After multiple adjustments we finally got the amplified product. The process is ongoing and we will be working on generating results on this modification post wiki freeze, as it was started late into the project, it could not be completed before that.

![  Unsuccessful PCR](https://static.igem.wiki/teams/5893/result/18.webp)
![ Successful PCR](https://static.igem.wiki/teams/5893/result/19.webp)

| Well | Sample |
| --- | --- |
| A1 | HiMedia 1kb DNA Ladder |
| A2 | PCR Product (set1) |

#### Learnings

Although amplification of RAD51 from genomic DNA proved difficult, this cycle highlighted the challenges of amplifying highly conserved or GC-rich genes. Future teams can overcome these issues by using synthetic gene synthesis or promoter engineering to upregulate native Rad51 expression, enabling efficient integration and testing under radiation stress. Due to time constraints and concurrent project tasks, this DBTL cycle will be continued post wiki freeze to make a mark for radiation-resistance optimization in yeast.

## DSUP

#### Overview

While RAD51 contributes to efficient repair of single- and double-strand DNA breaks through homologous recombination, additional protection may be provided by the tardigrade-derived protein DSUP (Damage Suppressor Gene). It is the unique element in Tardigrades that gives them the unique protection capabilities that they use to survive in extraterrestrial environments. Due to literature survey and the Part: BBa\_K2195000 from iGEM registry, we can confirm that DSUP has the ability to affect the survival rate in host organisms even while not being native to the organism. This was then followed up by optimization of the DSUP sequence for S. cerevisiae. We used the same GPD promoter and CYC1 terminator as in space, continuous expression of the gene is necessary.

#### Hypothesis

It was hypothesized that constitutive expression of DSUP in *S. cerevisiae* would improve radiation tolerance, enabling the yeast to better survive DNA damage induced by cosmic or UV radiation. Thus, yeast cells carrying this construct should demonstrate greater survivability and genomic stability compared to wild-type strains when exposed to simulated radiation stress.

#### Results

* The DSUP insert was successfully amplified and purified
* The clonal plasmids containing DSUP insert along with pRS426 plasmid were digested along with Tango buffer and Restriction enzymes Xba1 and Xho1 for 4.5hrs. After digestion, the insert from clonal plasmid containing DSUP was separated using Gel Electrophoresis
* Digestion of pRS426 plasmid initially faced issues that were resolved by switching to FD (Fast Digested) buffer.
* Ligation of DSUP into the pRS426GPD plasmid was completed before the wiki freeze. Transformation into *S. cerevisiae* and functional testing under radiation conditions could not be performed due to time and biosafety constraints. We planned to test its growth under radiation setup at a UV facility that we have access to.

![ Unsuccessful PCR](https://static.igem.wiki/teams/5893/result/20.webp)
![ Successful PCR](https://static.igem.wiki/teams/5893/result/21.webp)

| Lane | Sample |
| --- | --- |
| L1 | HiMedia High Range DNA Ladder |
| L2 | DSUP Digested Product (set 1) |
| L3 | DSUP Digested Product (set 2) |
| L4 | pRS426 Digested Product |
| L5 | pRS426 Plasmid |
| L6 | DSUP Plasmid (set 1) |
| L7 | DSUP Plasmid (set 2) |

| Lane | Sample |
| --- | --- |
| X1 | HiMedia High Range DNA Ladder |
| X2 | DSUP Digested Product (set 1) |
| X3 | pRS426 Improper Digested Product |
| X4 | DSUP Digested Product (set 2) |

#### Learnings

This cycle established a robust DSUP expression construct for yeast and highlighted challenges with plasmid digestion and insert purification. Future work will involve transforming the construct into *S. cerevisiae* and assessing radiation resistance. Successful expression of DSUP in yeast could provide a space-ready chassis for biomanufacturing and guide future teams in Space Village projects.

## Melanin

#### Overview

A serendipitous discovery made in our lab of melanin production while studying the nutritional requirements of *P. Vulgaris* regarding different amino acids. *P. Vulgaris*, when cultured in a medium in the presence of L-Tyrosine, It formed eumelanin via the Dopamine (catecholamine) biosynthetic pathway.

To utilize this finding and due to the fact that we thought that the current plan of radiation protection of our *S. cerevisiae* needed more protection from Galactic Cosmic Radiation, We integrated this eumelanin production pathway into our design for radiation protection, leveraging the well-established UV-shielding properties of eumelanin to enhance the resilience of our chassis against space radiation.

We finalized on dispersing eumelanin into a PVA sheet. This is because it is a sustainable, lightweight and inert material which shows great alignment with the idea.

#### Hypothesis

We hypothesized that embedding eumelanin into PVA sheets would provide substantial protection against UV and space-relevant radiation. The melanin is expected to absorb and reflect harmful wavelengths, preventing DNA damage and improving the survival of *S. cerevisiae*.

Additionally, we predicted that the combination of lightweight, porous PVA with melanin would create a shield suitable for space applications.

This material was hypothesized to be durable under prolonged radiation, maintaining structural integrity while preserving the viability of organisms underneath, making it a promising tool for space biomanufacturing and long-duration extraterrestrial experiments.

#### Results

Melanin was produced using the above-mentioned metabolic pathway present in *P. vulgaris. P. vulgaris* was cultured in a medium containing L-tyrosine and incubated for a week. The culture flask gradually turned darker until it became almost black, indicating the presence of eumelanin. This eumelanin was then extracted from the culture medium as documented in the protocols.

The dried powder was used to make a biopolymer with PVA by dissolving in a NaOH solution. It was then dried to make biopolymer-composite sheets of 10% w/v PVA
And 0.05 w/v% eumelanin.

These sheets were used for subsequent testing which included:

1. A preliminary visual test was done to evaluate the effectiveness of this eumelanin-containing biopolymer in UV shielding by placing it on a transilluminator and comparing it with a control sheet.

The results showed that the biopolymer-composite sheet is very effective at absorbing and reflecting UV, Visible light.

Next we moved on to UV-shielding quantification of the sheets:

Spread plates of *S. cerevisiae* were made of dilution 10^4, 300 microlitres of diluted culture was plated onto an agar medium, exposed to UV light in a LAF for 1 hour.

* Plate 1 - Sheet of PVA + Melanin + NaOH
* Plate 2 - Control sheet - PVA + NaOH
* Plate 3 - Uncovered plate outside the LAF as a control

* Control Plate (uncovered, outside LAF): (1.17 x 10^4 CFUs) Showed normal growth - Not exposed to UV radiation.
* Control Plate (PVA + NaOH only): (Negligible number of CFUs) Showed reduced colony growth, indicating that PVA alone provides minimal UV shielding.
* Test Plate (PVA + melanin + NaOH): (1.85 x 10^4 CFUs) Displayed significantly higher colony survival under UV compared to the PVA-only sheet - Indicating melanin based UV protection enhanced survival.

Additional observations - Control sheet showed visible burns and crumpling while no changes was seen the PVA+Melanin sheet

![](https://static.igem.wiki/teams/5893/engineering/final2/5m.avif)

#### Learnings

* Even distribution of melanin was critical; Sonication of the PVA+Melanin solution improved homogeneity in sheets.
* PVA+melanin sheets effectively absorb and reflect UV, improving yeast survival even under prolonged exposure.
* Higher CFU counts under the shielded condition may indicate epigenetic enhancement of stress resistance in S. cerevisiae.

Prospective experiments - A future collaboration with Dr. Joe Ninan (TIFR) is planned to test these sheets in high-altitude balloon experiments simulating cosmic and UV radiation. This will be conducted post wiki-freeze due to the time constraints.

All of these learnings solidified the usage of melanin as an extra layer of protection for the modified *S. Cerevisiae* inside the bioreactor. It proved the overall effectiveness of the melanin sheets in their application.

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