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Contribution

Introduction​

Our work involved two key areas: A comprehensive study of C. pepo, a vital food-producing plant, and the enhancement of genetic parts for S. cerevisiae, a widely used model organism. For C. pepo, we characterized its growth and explored its suitability as a model for phosphate deficiency tests. We also investigated the impact of copper concentrations on C. pepo's growth, essential for CUP1 induction. We developed a modified Hoagland solution protocol for testing biological phosphate fertilizers. Our research contributes to sustainable agriculture and improved food production. Simultaneously, we filled the gap in standardized genetic parts for S. cerevisiae by optimizing existing parts and introducing novel elements, thus simplifying cloning processes. These efforts empower researchers to act more freely when practicing synthetic biology. Our mission is to inspire future iGEM teams to continue advancing agriculture and genetic engineering.

Our work with C. pepo​

Our contribution to further teams involved extensive work with C. pepo, a plant that is highly important due to its ability to produce edible food. In comparison, Arabidopsis, another plant genus encompassing well established model organisms, does not possess the same significance in terms of food production. One of our major accomplishments was the characterization of C. pepo growth in general. This involved studying various aspects such as its growth rate while being supplied by Hoagland solution, and response to different phosphate conditions. By understanding these factors, we were able to provide valuable insights into optimizing the cultivation of C. pepo for testing with synthetic fertilizer systems.

Furthermore, we investigated whether C. pepo could be used as a suitable model for phosphate deficiency tests within a specific timeframe. Phosphate deficiency is a crucial issue in agriculture, and finding plants that can accurately reflect this condition is essential for developing effective solutions. Our findings indicate that C. pepo is sadly not suitable for such tests within the desired timespan of a classical iGEM project. Furthermore a higher number of plants might be better suited regarding statistical evaluation. Moreover, an additional month of growing might yield more clear results.

Another significant aspect of our work was determining if the copper concentrations required for reliable CUP1 induction would harm C. pepo growth. The CUP1 gene is commonly used as a marker in genetic studies and requires copper for its activation. However, it was crucial to identify the optimal copper concentration that would induce CUP1 without harming the overall growth and biomass of C. pepo. Our results suggested that certain copper concentrations may not statistically significantly affect the growth and biomass of C. pepo and may thus be used for copper induced ground fertilization testing systems.

In addition to our research on C. pepo, we also adapted a Hoagland solution protocol suitable for phosphate deficiency testing specifically aimed at biological phosphate fertilizers. Hoagland solution is a widely used nutrient solution for plant growth experiments 1, but modifications were needed to address the specific requirements of testing biological phosphate fertilizers in phosphate-deficient conditions. Our adapted protocol provides a standardized and reliable method for evaluating the effectiveness of these fertilizers in promoting plant growth under phosphate deficiency.

Overall, our work with C. pepo and the development of a suitable testing protocol for biological phosphate fertilizers can contribute significantly to further teams working in the field of plant biology and agriculture. By understanding the growth characteristics of C. pepo, its suitability for specific tests, and optimizing nutrient solutions for testing fertilizers, we have provided valuable knowledge and tools that can aid in the development of sustainable agricultural practices and improve food production.

S. cerevisiae: Our team made significant contributions to the synthetic biology community by enhancing the availability of essential genetic parts for Saccharomyces cerevisiae (S. cerevisiae), a widely used model organism. Despite the prevalence of S. cerevisiae as a model organism, we identified a notable lack of standardized biological parts in the iGEM registry, particularly in the realm of codon-optimized components. In response to this, we embarked on a mission to fill this void and empower future teams with a more comprehensive toolbox of genetic parts tailored for S. cerevisiae.

One of our primary endeavors was to optimize and refine existing genetic parts for S. cerevisiae, with a strong emphasis on codon optimization to enhance their synthesizing capability and minimize the use of rare codons. We meticulously curated and codon-optimized multiple parts that were originally documented in literature or were already present in the registry, thereby improving their compatibility with S. cerevisiae. Notable among these optimized parts were:

  1. meffRFP (BBa_K4706005): We subjected this reporter gene to rigorous codon optimization, ensuring that it would perform optimally within the genetic context of S. cerevisiae. This optimized reporter would prove invaluable in future experiments involving fluorescence-based assays and investigations.
  2. eechGFP1 (BBa_K4706008): Similar to meffRFP, we applied our codon optimization expertise to eechGFP1, another critical reporter gene. By enhancing its codon usage, we aimed to improve its expression levels and reliability as a reporter in S. cerevisiae studies. Both of these parts are sourced from the Open Reporters collection 2.

Additionally, we expanded the repertoire of functional genetic components for S. cerevisiae by introducing novel elements:

  1. SrpR-SV40NLS (BBa_K4706021): This component consists of a bacterial repressor with a nuclear localization signal (NLS) tag. Its efficiency has been demonstrated in literature, making it a valuable asset for regulating gene expression within S. cerevisiae.
  2. AtBAG6 (BBa_K4706007): As an alternative cell death protein to Bax, we introduced AtBAG6 as a novel genetic part. Its effectiveness in S. cerevisiae has been established in existing literature 3, providing researchers with a fresh tool for exploring cell death mechanisms in this model organism.

Furthermore, we recognized the importance of streamlined cloning processes in synthetic biology. To facilitate this, we designed a custom HO-Insert backbone (BBa_K4706000) that could be easily constructed through two cloning steps and subsequently linearized via PCR.

Additionally, we committed to easing the burden on future teams by codon optimizing potentially essential components in the registry:

  • BBa_K4706003: We included the binding site of SrpR as a standardized part in the registry. This addition ensures that future teams working with custom-regulated promoters will not have to retrieve it themselves from external sources, saving valuable time and effort.

In summary, our contributions concerning both C. pepo and S. cerevisiae aimed at significantly advancing the fields of plant biology and synthetic biology. We have expanded the genetic toolkit for S. cerevisiae, facilitating research in this model organism, while also providing valuable insights and tools for optimizing C. pepo cultivation and testing biological phosphate fertilizers. In view of these optimized tools and newly established protocols, we hope for future iGEM teams to pick up similar projects and continue our mission towards optimized nutrient application and enhanced yields in global agriculture.

  1. van Delden SH, Nazarideljou MJ, Marcelis LFM. Nutrient solutions for Arabidopsis thaliana: a study on nutrient solution composition in hydroponics systems. Plant Methods. 2020 May 18;16:72. doi: 10.1186/s13007-020-00606-4. PMID: 32612669; PMCID: PMC7324969.↩
  2. Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, et al. (2008) Diversity and Evolution of Coral Fluorescent Proteins. PLOS ONE 3(7): e2680. https://doi.org/10.1371/journal.pone.0002680↩
  3. Kang CH, Jung WY, Kang YH, Kim JY, Kim DG, Jeong JC, Baek DW, Jin JB, Lee JY, Kim MO, Chung WS, Mengiste T, Koiwa H, Kwak SS, Bahk JD, Lee SY, Nam JS, Yun DJ, Cho MJ. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death Differ. 2006 Jan;13(1):84-95. doi: 10.1038/sj.cdd.4401712. PMID: 16003391.↩