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| − | Understanding the genomic context of genes provides insights into their potential regulatory mechanisms and interactions with neighboring genes, which can be critical for their function. The genomic map in Figure | + | Understanding the genomic context of genes provides insights into their potential regulatory mechanisms and interactions with neighboring genes, which can be critical for their function. The genomic map in Figure illustrates the distribution of our selected genes across the nine chromosomes of <i>T. kok-saghyz</i>. |
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<img src="https://static.igem.wiki/teams/5088/registry/illustrations/consensus-sbol.png" width="900px" max-height:400px> | <img src="https://static.igem.wiki/teams/5088/registry/illustrations/consensus-sbol.png" width="900px" max-height:400px> | ||
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| − | <figcaption><p><b>Figure | + | <figcaption><p><b>Figure 7:</b> SBOL scheme of our <a href="http://parts.igem.org/Part:BBa_K5088677" target=blank_>Tarakate - consensus measurement construct [BBa_K5088677]</a>. |
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<img src="https://static.igem.wiki/teams/5088/registry/illustrations/protoplast-meassurement.png" width="450px" max-height:400px> | <img src="https://static.igem.wiki/teams/5088/registry/illustrations/protoplast-meassurement.png" width="450px" max-height:400px> | ||
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==Results== | ==Results== | ||
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| − | + | Cut-dip-budding (CDB) is an innovative plant transformation technique that involves immersing a cut segment of a plant in a transformation solution enriched with <i> Agrobacterium</i>. This process effectively facilitates the introduction of desired genetic material into the plant cells, enabling efficient genetic modification. The benefits of the CDB method include its ease of use and reduced labor requirements compared to traditional transformation techniques <a href="doi.org/10.1016/j.xinn.2022.100345">(Cao et al., 2023a)</a>. | |
| − | + | The CDB has been optimized for <i>Taraxacum</i> with a stable transformation method called "Extremely simplified cut-dip-budding," recently reported in „Extremely simplified cut-dip-budding method for genetic transformation and gene editing in Taraxacum kok-saghyz“ to regenerate stably transformed dandelion shoots within 14 days without requiring plant tissue culture or specialized equipment <a href="doi.org/10.59717/j.xinn-life.2023.100040">(Cao et al., 2023b)</a>. | |
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| − | + | This is an even faster stable transformation protocol than for model organism like <i>Arabidopsis thaliana</i>and easy to implement for iGEM teams, since it is not sterile and straight-forward. | |
</p> | </p> | ||
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| − | + | we selected two different promoter + 5' UTR constructs as a proof of concept to assess their ability to drive gene expression in stable transformations and across various tissues beyond leaves. We applied our rapid cut-dip protocol and successfully generated stably transformed dandelion roots. | |
| + | Fluorescence microscopy revealed clear signals of mCherry and eGFP in the transformed roots, confirming that the tubulin promoter + 5' UTR is functional in stable transformations. | ||
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| − | <a href="https://static.igem.wiki/teams/5088/wiki/images/results/ | + | <a href="https://static.igem.wiki/teams/5088/wiki/images/results/cdb-512-neg-new.webp" target="_blank"> |
| − | <img src="https://static.igem.wiki/teams/5088/wiki/images/results/ | + | <img src="https://static.igem.wiki/teams/5088/wiki/images/results/cdb-512-neg-new.webp" width="900px" max-height:400px> |
</a> | </a> | ||
| − | <figcaption><p><b>Figure</b>: | + | <figcaption><p><b>Figure 9</b>: Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control of T. kok-saghyz B) Measurement construct - Tubulin promoter+5'UTR from T. kok-saghyz [BBa_K5088512] tested in T. kok-saghyz.</figcaption> |
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Revision as of 03:37, 2 October 2024
Measurement construct - Tubulin promoter+5'UTR from T. kok-saghyz
This composite part serves as the measurement construct for the basic part BBa_K5088012 (Tubulin).
Contents
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Background
Motivation
Figure 1: Graphical abstract - from dandelion to natural rubber.
With our project Tarakate, we aim to explore the potential of the Russian dandelion (Taraxacum kok-saghyz) as a sustainable source of natural rubber. This plant, native to Kazakhstan, is unique for its ability to produce significant amounts of high-quality latex in its roots—a trait found in only a few species worldwide (1). Natural rubber is vital due to the global demand of roughly 15 million tons annually (2) and its application in more than 50,000 products (3), ranging from tires to medical supplies. As demand continues to rise, the limitations of traditional rubber sources, such as the rubber tree (Hevea brasiliensis), have become increasingly apparent. The production of rubber from H. brasiliensis has led to significant environmental and economic challenges, including deforestation of approximately four million hectares of rainforest, labor exploitation, and vulnerability to diseases like South American leaf blight (4, 5, 6). These issues, coupled with the geographic constraints of rubber tree cultivation—primarily restricted to tropical regions—underscore the urgent need for alternative rubber sources that can be cultivated in diverse climates and offer greater sustainability.
To harness the potential of T. kok-saghyz as a sustainable source of natural rubber, engineering priorities include optimizing biomass production and morphological traits, such as root architecture and seed size, to maximize yield, improve harvesting efficiency, and enhance overall agricultural practices for easier handling and processing. Besides targeting natural rubber production, there is also a focus on increasing the production of other valuable products like inulin (7), which can be used for the production of biofuel, and various bioactive compounds with potential applications in multiple industries (8). By developing alternative rubber sources like T. kok-saghyz, we aim to mitigate the environmental and economic impacts associated with traditional rubber production and contribute to a more sustainable future.
Identification
Based on the transcriptomics and functional genomics analysis we identified a selection of suitable genes for inclusion in our toolbox. These genes exhibited large dynamic range, low variance across different tissues, and were associated with essential cellular functions, confirming their suitability as sources of constitutive regulatory elements. One such gene that stood out was later identified as Tubulin.
Figure 2: Gene expression profile of all identified gene transcripts across 12 samples (with 3 biological replicates each), representing different tissue types and developmental stages. The y-axis displays the variance between samples, calculated using Variance Stabilizing Transformation (VST), while the x-axis shows the mean transcript expression in a regularized log scale. Dashed lines indicate the quantile filtering thresholds applied for both variance and mean expression. The purple-shaded region highlights the area of interest, containing 764 genes that were selected for further analysis. The gene Tubulin, corresponding to this regulatory part, is highlighted within this region.
To better understand the potential regulatory utility of Tubulin, we focused on its specific expression pattern across various tissues and developmental stages. Although Tubulin is not among the highest-expressing genes, it exhibits remarkably low variance, which is a crucial feature for Plant SynBio, where predictable and stable gene expression is often more valuable than sheer expression strength.
Figure 3: Normalized counts of Tubulin across various tissue types and developmental stages using DESeq2’s median of ratios normalization method. Each bar shows the mean normalized count for Tubulin across the samples, with error bars indicating the standard deviation. The dots represent individual biological replicates for each sample.
To quantify $nicknames expression stability more precisely, we summarized key statistical measures derived from both the VST and regularized log transformations.
Genetic Context and Gene Structure
Figure 4: Genomic map showing the distribution of selected genes across nine chromosomes (Chr 1 to Chr 9). The scale on the left represents chromosome lengths in megabases (Mb). The positions of the genes are indicated along the chromosomes, with the gene Tubulin emphasized in bold.
Understanding the genomic context of genes provides insights into their potential regulatory mechanisms and interactions with neighboring genes, which can be critical for their function. The genomic map in Figure illustrates the distribution of our selected genes across the nine chromosomes of T. kok-saghyz.
Corresponding Protein and Function
Figure 5: Hierarchical overview of Gene Ontology (GO) terms associated with the Tubulin protein, as predicted by eggNOG during the annotation process. The GO terms are organized into categories representing Molecular Function, Biological Process, and Cellular Component, showing the relationships and hierarchies between them. This visualization was generated using the EBI"s QuickGO tool and provides a detailed breakdown of the functional annotations associated with the Tubulin gene.
The hierarchical GO view included more detail than needed, making it difficult to digest. Inspired by the GO ribbon on UniProt, which offers a simplified summary of gene functions, we sought a similar visualization for our gene. However, no tools were available to create such visualizations for non-model organisms.
To address this, we developed our own tool to map the GO terms to the "plant slimset", a condensed version of GO terms specifically tailored to highlight key functional categories in plants. This approach enabled us to create a ribbon diagram summarizing the gene"s GO annotations across Biological Process, Molecular Function, and Cellular Component.
Figure 6: Ribbon diagram summarizing GO annotations for Tubulin. The diagram is divided into three sections—Biological Process, Molecular Function, and Cellular Component—each representing high-level GO terms. The color gradient from white to green indicates the number of corresponding annotations, with deeper shades representing higher values.
Testing & Measurement
Dual Fluorescence Reporter Assay
Experiments in plant biology are often susceptible to high variability, with factors like transformation efficiency, cell viability, and environmental conditions contributing to noisy results.
To mitigate these issues, we employed a dual fluorescence reporter assay. In this setup, both the target and reference proteins are expressed from the same plasmid, allowing for precise and reliable characterization.
Using a second, constant fluorescence reporter as an internal reference allows us to normalize the readout, ensuring that observed fluorescence accurately reflect the activity of the regulatory elements under study, independent of external factors.
Figure 7: SBOL scheme of our Tarakate - consensus measurement construct [BBa_K5088677].
Choice of Reporter Genes
For our initial round of testing, we evaluated a construct design using the plant GFP from the iGEM distribution kit as a reporter system.
In our initial experiments, we were unable to detect any fluorescence in dandelion leaf infiltrations or protoplast transformations. To troubleshoot, we tested the construct in tobacco leaf infiltrations. This process required several rounds of protocol optimization before we finally achieved positive GFP expression in tobacco leaves. After further adjustments, we also obtained very low GFP signals in dandelion protoplasts.
Despite these optimizations, the signal strength remained weak, registering only about three times above background levels. This indicated that the system was still not sensitive enough for effective promoter characterization. The avGFP from the iGEM distribution kit, an older and less bright variant, likely contributed to the low signal observed.
To address this, we switched to a more advanced GFP variant, eGFP, known for its higher brightness. Moreover we did extensive literature research and found out that Agrobacterium is able to use some plant promoters, leading to fluorescence that would be indistinguishable from the plant tissue"s expression.
Therefore we decided to introduce the potato ST-LS1 intron into the eGFP coding sequence to prevent Agrobacterium from expressing GFP, as this has been described as a suitable strategy for that problem.
A further challenge we encountered was the variability in transformation efficiency during Agrobacterium-mediated leaf infiltration and protoplast transformation. This variability made it difficult to perform quantitative measurements of the individual regulatory parts across biological replicates. To overcome this issue, we adopted the previously reported ratiometric approach by incorporating a second reporter gene in each construct. This reference reporter is located on the same plasmid as the GFP to minimize noise and provide a reliable normalization standard for more accurate quantitative analysis. To read more about the optimization of our reporter construct visit our engineering page
Protoplast Measurement
Protocol for Protoplast Measurement
- All ratiometric protoplast measurements were performed using a plate reader (TECAN INFINITE M PLEX)
- Measure autofluorescence (Ex 472 nm, Em 685 nm)
- Measure mCherry (Ex 570 nm, Em 610 nm)
- Measure eGFP (Ex 480 nm, Em 515 nm)
Figure 8: Schematic illustration of protoplast assay platereader measurement.
Results
Cut-dip-budding (CDB) is an innovative plant transformation technique that involves immersing a cut segment of a plant in a transformation solution enriched with Agrobacterium. This process effectively facilitates the introduction of desired genetic material into the plant cells, enabling efficient genetic modification. The benefits of the CDB method include its ease of use and reduced labor requirements compared to traditional transformation techniques (Cao et al., 2023a). The CDB has been optimized for Taraxacum with a stable transformation method called "Extremely simplified cut-dip-budding," recently reported in „Extremely simplified cut-dip-budding method for genetic transformation and gene editing in Taraxacum kok-saghyz“ to regenerate stably transformed dandelion shoots within 14 days without requiring plant tissue culture or specialized equipment (Cao et al., 2023b).
This is an even faster stable transformation protocol than for model organism like Arabidopsis thalianaand easy to implement for iGEM teams, since it is not sterile and straight-forward.
we selected two different promoter + 5' UTR constructs as a proof of concept to assess their ability to drive gene expression in stable transformations and across various tissues beyond leaves. We applied our rapid cut-dip protocol and successfully generated stably transformed dandelion roots. Fluorescence microscopy revealed clear signals of mCherry and eGFP in the transformed roots, confirming that the tubulin promoter + 5' UTR is functional in stable transformations.
Figure 9: Microscopy image of Cut dip budding transformed Taraxacum roots. Each subfigure is subdivided into four panels, where BF stands for brightfield and AF for autofluorescence. Shown below is the according mCherry and eGFP signal of the same root. A) Negative control of T. kok-saghyz B) Measurement construct - Tubulin promoter+5'UTR from T. kok-saghyz [BBa_K5088512] tested in T. kok-saghyz.
The Dandelion Toolbox
Our project aimed to advance the genetic engineering of dandelions by developing a robust set of constitutive regulatory parts. Using a transcriptomic approach, we identified 40 endogenous elements. To ensure precise and reliable testing, we constructed a ratiometric measurement system, enabling effective and quantitative characterization of these parts.
We employed three distinct plant transformation methods to test and validate the functionality of the regulatory elements. Through rigorous testing, we successfully characterized 23 out of the initial 40 elements, resulting in a comprehensive collection of standardized dandelion parts. This well-characterized suite of parts is designed to streamline future complex genetic engineering projects.
By providing these standardized tools, our project significantly lowers the barriers for researchers and iGEM teams, making Taraxacum kok-saghyz a more accessible and versatile chassis for plant synthetic biology. Ultimately, our work contributes to enhancing dandelion as a model organism and supporting sustainable natural rubber production.
Overview
| Part Identifier | Part Type | Nickname | Part Description |
|---|---|---|---|
| BBa_K5088001 | Promoter + 5'UTR | P_RPL28 | Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088006 | Promoter + 5'UTR | P_FKBP4_5 | FK506-binding protein 4/5 - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088007 | Promoter + 5'UTR | P_CLTC | Clathrin - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088008 | Promoter + 5'UTR | P_RPL31 | Large subunit ribosomal protein L31e - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088012 | Promoter + 5'UTR | P_Tubulin | Tubulin - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088013 | Promoter + 5'UTR | P_EIF5A | Translation initiation factor 5A - Promoter+5'UTR from T. kok-saghyz |
| BBa_K5088102 | 3'UTR | T_PTI1 | Protein tyrosine kinase - 3'UTR from T. kok-saghyz |
| BBa_K5088103 | 3'UTR | T_RPL28 | Large subunit ribosomal protein L28e - 3'UTR from T. kok-saghyz |
| BBa_K5088104 | 3'UTR | T_EPS15 | Epidermal growth factor receptor substrate 15 - 3'UTR from T. kok-saghyz |
| BBa_K5088105 | 3'UTR | T_GSK3B | Glycogen synthase kinase 3 - 3'UTR from T. kok-saghyz |
| BBa_K5088106 | 3'UTR | T_MGRN1 | E3 ubiquitin-protein ligase - 3'UTR from T. kok-saghyz |
| BBa_K5088107 | 3'UTR | T_RPL35A | Large subunit ribosomal protein L35Ae - 3'UTR from T. kok-saghyz |
| BBa_K5088108 | 3'UTR | T_betB | Betaine-aldehyde dehydrogenase - 3'UTR from T. kok-saghyz |
| BBa_K5088109 | 3'UTR | T_pgm | Phosphoglucomutase - 3'UTR from T. kok-saghyz |
| BBa_K5088110 | 3'UTR | T_ATP-synt | ATPase subunit gamma - 3'UTR from T. kok-saghyz |
| BBa_K5088111 | 3'UTR | T_EIF3B | Translation initiation factor 3 subunit B - 3'UTR from T. kok-saghyz |
| BBa_K5088112 | 3'UTR | T_RPL31 | Large subunit ribosomal protein L31e - 3'UTR from T. kok-saghyz |
| BBa_K5088113 | 3'UTR | T_TM9SF2_4 | Transmembrane 9 superfamily member 2/4 - 3'UTR from T. kok-saghyz |
| BBa_K5088114 | 3'UTR | T_CUL1 | Cullin - 3'UTR from T. kok-saghyz |
| BBa_K5088115 | 3'UTR | T_PSMB6 | 20S proteasome subunit beta 1 - 3'UTR from T. kok-saghyz |
| BBa_K5088116 | 3'UTR | T_RPSA | Small subunit ribosomal protein SAe - 3'UTR from T. kok-saghyz |
| BBa_K5088117 | 3'UTR | T_VPS4 | Vacuolar protein-sorting-associated protein 4 - 3'UTR from T. kok-saghyz |
| BBa_K5088118 | 3'UTR | T_EIF2S3 | Translation initiation factor 2 subunit 3 - 3'UTR from T. kok-saghyz |
Dandelion Handbook
By creating a suite of genetic tools and transformation methods, and sharing them through our Dandelion Handbook, we believe that dandelions can serve as an excellent chassis for numerous applications. We aim to inspire future iGEM teams to harness the unique properties of dandelions for a variety of promising projects.
Dandelions have demonstrated their versatility, being used as a coffee alternative and in various food applications such as salads, wine, and honey. Additionally, their ability to naturally hyperaccumulate environmental pollutants, including heavy metals, highlights their potential for bioremediation applications.
By equipping future iGEM teams with these resources, we aspire to unlock the full potential of dandelions, paving the way for sustainable and diverse synthetic biology applications.
Click here to look at our Dandelion Handbook
.References
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(2) MRC, Malaysian Rubber Council (MRC), MRC Official Website. https://www.myrubbercouncil.com/.
(3) Cherian, S., Ryu, S. B., & Cornish, K. (2019). Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects. In Plant Biotechnology Journal (Vol. 17, Issue 11, pp. 2041–2061). Wiley. https://doi.org/10.1111/pbi.13181
(4) R. Lieberei, South American Leaf Blight of the Rubber Tree (Hevea spp.): New Steps in Plant Domestication using Physiological Features and Molecular Markers. Ann. Bot. 100, 1125–1142 (2007).
(5) T. S. Suryanarayanan, J. L. Azevedo, From forest to plantation: a brief history of the rubber tree. Indian J. Hist. Sci. 58, 74–78 (2023).
(6) Y. Wang, P. M. Hollingsworth, D. Zhai, C. D. West, J. M. H. Green, H. Chen, K. Hurni, Y. Su, E. Warren-Thomas, J. Xu, A. Ahrends, High-resolution maps show that rubber causes substantial deforestation. Nature 623, 340–346 (2023).
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(8) S. Piccolella, C. Sirignano, S. Pacifico, E. Fantini, L. Daddiego, P. Facella, L. Lopez, O. T. Scafati, F. Panara, D. Rigano, Beyond natural rubber: Taraxacum kok-saghyz and Taraxacum brevicorniculatum as sources of bioactive compounds. Ind. Crops Prod. 195, 116446 (2023).
(9) J. Collins-Silva, A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish, D. Shintani, Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism. Phytochemistry 79, 46–56 (2012).
(10) X. Cao, H. Xie, M. Song, J. Lu, P. Ma, B. Huang, M. Wang, Y. Tian, F. Chen, J. Peng, Z. Lang, G. Li, J.-K. Zhu, Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture. The Innovation 4, 100345 (2023).
(11) A. Stolze, A. Wanke, N. van Deenen, R. Geyer, D. Prüfer, C. Schulze Gronover, Development of rubber-enriched dandelion varieties by metabolic engineering of the inulin pathway. Plant Biotechnol. J. 15, 740–753 (2017).
(12) N. van Deenen, K. Unland, D. Prüfer, C. Schulze Gronover, Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber. Molecules 24, 2703 (2019).
(13) S. M. Wolters, V. A. Benninghaus, K.-U. Roelfs, N. van Deenen, R. M. Twyman, D. Prüfer, C. Schulze Gronover, Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis. Front. Plant Sci. 14 (2023).
(14) V. A. Benninghaus, N. van Deenen, B. Müller, K.-U. Roelfs, I. Lassowskat, I. Finkemeier, D. Prüfer, C. Schulze Gronover, Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology. J. Exp. Bot. 71, 1278–1293 (2020).
(15) I. Ganesh, S. C. Choi, S. W. Bae, J.-C. Park, S. B. Ryu, Heterologous activation of the Hevea PEP16 promoter in the rubber-producing laticiferous tissues of Taraxacum kok-saghyz. Sci. Rep. 10, 10844 (2020).
(16) A. Wieghaus, D. Prüfer, C. S. Gronover, Loss of function mutation of the Rapid Alkalinization Factor (RALF1)-like peptide in the dandelion Taraxacum koksaghyz entails a high-biomass taproot phenotype. PLOS ONE 14, e0217454 (2019).
(17) E. Niephaus, B. Müller, N. van Deenen, I. Lassowskat, M. Bonin, I. Finkemeier, D. Prüfer, C. Schulze Gronover, Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis-prenyltransferase-like 1 protein. Plant J. 100, 591–609 (2019).
(18) A. Zhou u. a., „A Suite of Constitutive Promoters for Tuning Gene Expression in Plants“, ACS Synth. Biol., Bd. 12, Nr. 5, S. 1533–1545, Mai 2023, doi: 10.1021/acssynbio.3c00075.
(19) T. Lin u. a., „Extensive sequence divergence between the reference genomes of Taraxacum kok-saghyz and Taraxacum mongolicum“, Sci. China Life Sci., Bd. 65, Nr. 3, S. 515–528, März 2022, doi: 10.1007/s11427-021-2033-2.
(20) T. Lin u. a., „Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis“, Natl. Sci. Rev., Bd. 5, Nr. 1, S. 78–87, Jan. 2018, doi: 10.1093/nsr/nwx101.
(21) G. Vancanneyt, R. Schmidt, A. O’Connor-Sanchez, L. Willmitzer, und M. Rocha-Sosa, „Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation“, Mol. Gen. Genet. MGG, Bd. 220, Nr. 2, S. 245–250, Jan. 1990, doi: 10.1007/BF00260489.
(22) A. F. Ibrahim, J. A. Watters, G. P. Clark, C. J. Thomas, J. W. Brown, und C. G. Simpson, „Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5’ splice site“, Mol. Genet. Genomics MGG, Bd. 265, Nr. 3, S. 455–460, Mai 2001, doi: 10.1007/s004380000433.