Part:BBa_K5088614

Measurement construct - 3'UTR RPSA from T. kok-saghyz

This composite part serves as the measurement construct for the basic part BBa_K5088116 (RPSA).

Assembly Compatibility:

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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 RPSA.

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 RPSA, corresponding to this regulatory part, is highlighted within this region.

To better understand the potential regulatory utility of RPSA, we focused on its specific expression pattern across various tissues and developmental stages. Although RPSA 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 RPSA across various tissue types and developmental stages using DESeq2’s median of ratios normalization method. Each bar shows the mean normalized count for RPSA 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 RPSA 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 X 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 RPSA 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 RPSA 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 RPSA. 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 13: 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

Transient Transformation

The characterization of genetic parts is crucial for understanding their functionality and behavior within a host organism. However, this process presents significant challenges, particularly when working with plants, due to the time-intensive nature of stable transformation. Stable transformation involves the integration of foreign DNA into the host genome, ensuring that the genetic modifications are inherited by subsequent generations. While this approach is crucial for characterizing the parts in their intended context, it also comes with certain drawbacks.

The process of developing stable transgenic lines is time-consuming and resource-intensive due to the need for multiple homozygous lines to ensure consistent and detectable transgene expression, which is also influenced by local genetic context. This can be especially challenging for iGEM projects. To address these challenges, transient transformation offers a faster and more efficient alternative for the rapid characterization of genetic parts.

Transient transformation enables temporary expression of a reporter construct without the need for stable integration into the host genome, significantly reducing the time required to obtain results and allowing for faster iteration and optimization of genetic constructs. Techniques such as leaf infiltration and protoplast assays are already standard in plant biology when working with model organisms like Nicotiana benthamiana. We opted to use these methods in Taraxacum and optimize these methods in order to test genetic constructs in an easier fashion.

Leaf Infiltration

Leaf infiltration is a technique used to introduce foreign DNA, proteins, or molecules into plant tissues, enabling transient gene expression without stable transformation and commonly used in Nicotiana benthamiana. It involves infiltrating a solution to the leaf surface, allowing for rapid, non-permanent expression of target genes. This method offers several benefits, including fast results, cost-effectiveness by bypassing the need for transgenic plants, and versatility in testing gene constructs or protein interactions.

We utilized the method outlined in “Genetic transformation technologies for the common dandelion, Taraxacum officinale.”

Figure 14: Workflow illustration for leaf infiltration in Taraxacum species.

Experiments were conducted using Agrobacterium rhizogenes K599 and the RNA silencing suppressor P19 in Agrobacterium tumefaciens GV3101. Both strains were cultured to an optical density (OD600) of 0.6–0.8, harvested by centrifugation, and washed twice with infiltration buffer to remove proteins from Agrobacterium that stress the plants and hinder the transformation process. The infiltration solution was adjusted to an OD600 of 0.1 in infiltration buffer, supplemented with 10 µM acetosyringone, and incubated in the dark for 3 hours.

Three- to four-week-old Taraxacum plants were infiltrated by injecting the solution into the abaxial surface of the leaves using a 1 mL syringe without a needle. Four days post-infiltration, the plants were screened for gene expression to assess transformation efficiency (Figure 2). Read more detailed protocol here: Leaf infiltration protocol.

Additionally, we modified the protocol by heavily watering the plants and covering it with a hood for 3 hours prior to infiltration to facilitate the opening of leaf stomata, thus enhancing the ease of infiltration.

Protoplast Transformation

Protoplast transformation introduces foreign DNA into plant cells without cell walls, enabling rapid gene expression and precise genetic modification. It offers high efficiency and allows researchers to study cellular processes in a controlled environment, making it a key tool in functional genomics. Furthermore, it is possible to regenerate whole transgenic plants from transformed protoplasts by culturing them in a suitable regeneration medium supplemented with specific hormones that promote cell division, callus formation, and shoot regeneration.

As a starting point, we received guidance from Prof Dr Dirk Prüfer, who provided a method for efficient protoplast isolation. For protoplast transfection, we employed techniques described in the publications “Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis” and “Genetic transformation technologies for the common dandelion, Taraxacum officinale.” We further refined these methods through multiple cycles of optimization to adapt them for our system.

Figure 15: Illustration of Taraxacum protoplast transformation >

Our experiment utilized four-week-old Taraxacum plants, where the third to fifth leaves were gently scratched on the abaxial side to remove the outer cell layer. The leaves were then submerged in 10 mL of enzyme solution (1% cellulase R-10 and 0,5% macerozyme R-10) and incubated in the dark at 30°C for 4 hours, with gentle swirling indicating protoplast release. The mixture was filtered through a 100 µm cell strainer and centrifuged, resulting in floating protoplasts, enzyme solution, and cell debris. The debris and enzyme solution were removed, and the protoplasts were washed and counted using a hemacytometer.

For transfection, polyethylene glycol (PEG) was used with 20 µg of plasmid DNA and 20,000 protoplasts. After a 15-minute dark incubation, the mixture was transferred to a protoplast-optimized solution. Gene expression in the transformed protoplasts was observed between 15 and 24 hours post-transfection (Figure 4).

Read more detailed protocol here: Protoplast transformation protocol.

We determined that the number of protoplasts increased with extended incubation times of Taraxacum leaves in the enzyme solution. However, a 4-hour incubation was optimal, as longer durations led to reduced protoplast viability. To minimize damage from protoplast proteases, we maintained the protoplasts on ice during all washing steps. Additionally, we observed higher transformation efficiency when the protoplasts were incubated in the dark during the transfection process, likely due to reduced stress and activation of defense mechanisms in the protoplasts.

Measurement Setup

Leaf Disk Measurement

A challenge of leaf infiltration and protoplast assays is the variability in transformation efficiency, complicating quantitative measurements across biological replicates. To solve this, we adopted a ratiometric approach, incorporating mCherry as a secondary reporter driven by the Arabidopsis ubiquitin promoter, allowing for normalization and more accurate quantification (2).

Materials

1. Plate reader
2. 96-well or 384-well plate black, clear flat bottom
3. Forceps
4. 0.7 mm cork borer

Protocol for Leaf Disk Measurement

1. Fill every well with 10µl ddH2O
2. Punch out the leaf disks using a cork borer. Then, place them with a forceps in the 96-well plate abaxial side down
3. Make sure to close the lid in between to prevent dehydration
4. We performed all ratiometric leaf disk assays in a plate reader (BMG labtech, CLARIOstar Plus)
5. Settings for eGFP (Ex: 472nm ± 6nm, Em: 515 ± 6nm)
6. Settings for mCherry (Ex: 570nm ± 6nm, Em: 610nm ± 6nm)

Figure 16: Schematic representation of the ratiometric measurement system used for the characterization of regulatory parts in T. kok-saghyz. The process begins with (1) the preparation of the infiltration solution, followed by (2) leaf infiltration using a syringe. After (3) a 4-day incubation period, leaf disks are (4) punched out and placed in a 96-well plate. (5) The samples are measured using a plate reader, and (6) ratiometric analysis is conducted, comparing eGFP and mCherry signals for quantitative characterization of the regulatory parts.

Protoplast Measurement

Protocol for Protoplast Measurement

1. All ratiometric protoplast measurements were performed using a plate reader (TECAN INFINITE M PLEX)
2. Measure autofluorescence (Ex 472 nm, Em 685 nm)
3. Measure mCherry (Ex 570 nm, Em 610 nm)
4. Measure eGFP (Ex 480 nm, Em 515 nm)

Figure 17: Schematic illustration of protoplast assay platereader measurement.

Results

GFP and mCherry signals for most 3' UTR constructs in both TKS and TO, can successfully be detected via plate reader measurements of the transiently transformed protoplasts demonstrating reliable expression. These results highlight the robustness of our ratiometric approach in characterizing 3'UTR elements via transient protoplast transformation.

Figure: Protoplast assay using Taraxacum officinale. Shown are (A) eGFP expression, (B) mCherry expression and (C) ratiometric between both reporters.

Figure: Protoplast assay using Taraxacum kok-sagyhz. Shown are (A) eGFP expression, (B) mCherry expression and (C) ratiometric between both reporters.

Furthermore, similar to those protoplast results, we successfully detected reporter gene signals for both mCherry and eGFP in most of the 3' UTR constructs during tobacco leaf infiltrations, indicating that the design of these regulatory elements is generally viable.

Figure: Leaf infiltration assay using Nicotiana benthamiana. Shown are (A) eGFP expression, (B) mCherry expression and (C) ratiometric between both reporters.

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

Table 1: List of functioning T. kok-saghyz endogenous regulatory elements we've characterized in our project

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

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