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.
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.
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.
It was hypothesized that the deletion of GAT1 gene which is a key activator in the Nitrogen Catabolite repressor system, would:
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.
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.
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.
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.
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:
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
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.
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.
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:
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.
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.
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.
Primers were designed for PCR amplification of the RAD51 gene using genomic DNA extracted from wild-type S. cerevisiae.
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.
| Well | Sample |
|---|---|
| A1 | HiMedia 1kb DNA Ladder |
| A2 | PCR Product (set1) |
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.
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.
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.
| 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) |
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.
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.
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.
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:
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.
Additional observations - Control sheet showed visible burns and crumpling while no changes was seen the PVA+Melanin sheet
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.