Part:BBa_K5088677
Tarakate - Consensus measurement construct
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The Challenge
To achieve our key goal of a genetic toolbox for dandelion synthetic biology, we set out to create a system for characterization of regulatory elements. One of the fundamental challenges we encountered was determining how to effectively test them in Taraxacum kok-saghyz (Russian dandelion), a non-model species. Part characterization in plants is inherently difficult due to long engineering cycles, limited testing throughput, and the complexity of gene expression across different tissues and developmental stages. While fast part characterization systems exist, they are largely limited to a few model plant species. Non-model species like dandelion often lack established protocols for transient testing, further complicating the process. As a result, these species are left with relatively few regulatory parts available for plant engineering.
To create our toolbox, we required a characterization plasmid where the selected regulatory elements could drive the expression of a reporter gene for measurement. Additionally, it should contain the necessary elements for transformation of the construct in plants.
Engineering Cycles
Initial Considerations
During the creation of a part characterization construct, the choice of reporter gene is exceptionally important, as it will define the method for measurement and its precision. For initial protocol optimization, we utilized the RUBY system, which offers the main advantage of easy visualization of gene expression across tissues, without the need for fluorescence microscopy. Instead, it works by producing a red-colored secondary metabolite visible to the naked eye. However, while it is a great maker for qualitative results, its reliance on an enzyme cascade to produce the pigment makes it unsuitable to quantitatively measure gene expression. To address this limitation, we opted to use a fluorescent reporter system, which allows for more precise quantification of the regulatory parts, providing a clearer measure of their performance.
The process of building plasmids for each one of the parts involves a large amount of cloning work. Therefore, we opted to use the MoClo system. The modularity of this cloning standard and its use of standardized genetic elements allows the assembly of a large set of constructs in a short amount of time. Additionally, the overhangs used in our constructs followed the Phytobrick standard, which is commonly used in the plant synbio and the iGEM communities. These early design choices ensure that all regulatory parts in our toolbox can be easily used by future researches and iGEM teams. It should be highlighted that without the adoption of MoClo, we wouldn’t have been able to build all the test constructs within the timeline of iGEM, demonstrating the advantages of standardization.
Cycle 1 - The First Proof of Concept
Figure 1: SBOL scheme of Tarakate - Test construct GFP [BBa_K5088675]
In our first design, the expression system used the GFP from the plant collection in the iGEM distribution kit. The expression was driven by the 35S CaMV promoter, TMV 5' UTR, and 35S 3' UTR. This system is widely used for constitutive overexpression of transgenes in various plant species, including model organisms and economically important crops.
Initially, 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.
Figure 2: Successful expression of avGFP in protoplasts of Taraxacum officinale using Tarakate - Test construct GFP [BBa_K5088675]
Figure 3: Successful expression of avGFP in N. benthamiana leaf disk assay using Tarakate - Test construct GFP [BBa_K5088675]
Figure 4: Successful expression of avGFP in roots of T. kok-saghyz using the CDB protocol and Tarakate - Test construct GFP [BBa_K5088675] A) Brightfield view of a transformed root of T. kok-saghyz B) The same root imaged under UV light.
These results led us to suspect that the GFP being used was not bright enough for the assay. Indeed, we found that the variant shipped with the distribution kit (avGFP) is the original version isolated from Aequorea victoria in 1962 (Shimomura et al., 1962). Using this old and dimmer fluorescent protein likely contributed to the low signal observed.
To address this issue, we switched the reporter to eGFP, a modern and enhanced variety, known for its higher brightness. Moreover, we found through extensive literature research that transcription in Agrobacterium can be initiated by certain plant promoters, leading to fluorescence that would result in false positives in the plant tissue (false positives). Therefore, we decided to introduce the potato ST-LS1 intron into the eGFP coding sequence to prevent Agrobacterium from expressing it, as this has been described as a suitable strategy to fix that problem (Brophy et al., 2022).
A further challenge we encountered was the variability in transformation efficiency during Agrobacterium-mediated leaf infiltration and protoplast transformation. This 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 (Schaumberg et al., 2016). This reference reporter is located on the same plasmid as the GFP, and its expression can be used as a reliable normalization standard that reduces noise and provides more accurate quantitative analysis.
Cycle 2 - Switching to a Ratiometric Measurement
After the lessons learned in the first round of testing, we implemented several key changes in our new design:
- Moved away from avGFP to the brighter eGFP for improved signal strength.
- Introduced an intron into the coding sequence of eGFP to prevent expression in Agrobacterium.
- Introduced a secondary reporter (mCherry driven by the Arabidopsis ubiquitin promoter) for ratiometric normalization.
Figure 5:SBOL scheme of this construct, BBa_K5088677
To confirm the viability of the changes made, we transformed the new constructs in tobacco as a proof of concept.
We could show that the brightness of eGFP was substantially higher when compared to the avGFP used before. Additionally, we quantified mCherry fluorescence and calculated the GFP/mCherry ratio, demonstrating the viability of the ratiometric measurement.
Figure 6:Comparison between Tarakate - Test construct GFP [BBa_K5088675] and Tarakate - Consensus measurement construct [BBa_K5088677]. The control represents non infiltrated leaf disk of N. benthamiana, K599 show leaf disks infiltrated with A. rhizogenes K599 and A. tumefaciens GV3101 P19 as additional negative controls. A) avGFP expression [Ex: 408nm ± 12nm; Em: 515nm ± 12nm] B) mCherry expression [Ex: 570nm ± 12nm; Em: 610nm ± 12nm] C) eGFP expression [Ex: 472nm ± 12nm; Em: 515nm ± 12nm].
Once the normalization system had been proven in tobacco, we proceeded to evaluate the updated version of our plasmid in T. kok-saghyz protoplasts. We characterized 35 different constructs, each varying in their promoter + 5’ untranslated region (UTR) or 3’ UTR elements. The experiments successfully detected both eGFP and mCherry signals, demonstrating that this approach is also viable in non-model plants like dandelion. Most important, the mCherry normalization cassette, driven by the Arabidopsis ubiquitin promoter, functioned effectively in dandelion, validating its use for future experiments.
Figure 7:Measurement of Promoter + 5'UTR parts in protoplast assay of T. kok-saghyz. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
Figure 8:Measurement of 3'UTR parts in protoplast assay of T. kok-saghyz. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
Figure 9:Measurement of Promoter + 5'UTR parts in protoplast assay of T. officinale. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
Figure 10:Measurement of 3'UTR parts in protoplast assay of T. officinale. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
While the mCherry signal was detected in nearly all constructs, eGFP expression was only observed in the constructs containing different endogenous T. kok-saghyz 3'UTRs. Surprisingly, no signal was detected when using the 35S 3'UTR, which is widely adopted in Plant Synthetic Biology. As a result, we identified 13 alternative 3’ UTRs that led to significantly higher reporter gene expression compared to the 35S 3'UTR.
Additionally, we found that promoters + 5'UTRs other than the 35S promoter did not produce sufficient gene expression in our protoplast testing system.
From our experiments, we identified that gene expression was significantly enhanced by several 3’UTRs, which we selected to incorporate into the next version of the ratiometric construct. However, before settling on a new design, we needed to investigate whether the performance of the promoter + 5’UTRs could be optimized in a different testing system. This approach would allow us to determine if a design with stronger or more consistent expression could be found, providing valuable insights before moving forward with an improved construct design.
Cycle 3 - Troubleshooting via Another Plant Chassis
To troubleshoot the results obtained from the different promoter + 5'UTR constructs, we reverted to tobacco leaf infiltrations as a testing system, where expression levels are generally higher. This experiment provided a more robust environment for assessing the performance of these parts.
For the transient leaf infiltration in tobacco all promoter + 5'UTR or 3'UTR elements were used again to identify any potential candidates for testing in dandelion.
Figure 11:Measurement of Promoter + 5'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
Figure 12:Measurement of 3'UTR parts in leaf disk assay of N. benthamiana. Graphs show expression of A) eGFP, B) mCherry. C) Shows the normalized ratio of log of eGFP divided by log of mCherry signal.
Similar to our previous protoplast results, we successfully detected reporter gene signals for both mCherry and eGFP in most of the 3'UTR constructs during tobacco leaf infiltrations. Notably, we also observed eGFP expression in some of the Promoter + 5'UTR constructs, indicating that the design of these regulatory elements is generally viable. These findings suggest that while the constructs themselves are functional, improvements in our testing system are needed to consistently capture their full potential across different environments.
Moreover, we demonstrated that our endogenous dandelion regulatory elements are not only functional in Taraxacum kok-saghyz, but are also broadly applicable to other plant chassis. This expands their utility, making them a versatile resource for future iGEM teams working on any other plant species. By providing regulatory elements with cross-species functionality, we are enabling more flexible and efficient plant engineering efforts across different contexts.
Cycle 4 - Stable Dandelion Transformation
Following the promising results from our tobacco test system, we aimed to determine whether the lack of expression from our promoter + 5'UTR parts in dandelion was due to the promoters themselves or if further optimization of our transient testing system.
To investigate this, 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. This result indicates that our regulatory element designs are effective and suggests that optimizing our transient testing system is necessary to obtain reliable results for the remaining promoter + 5'UTR constructs.
Figure 13: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 B) Tarakate - Consensus measurement construct [BBa_K5088677] in T. kok-saghyz C) Tarakate - Consensus measurement construct [BBa_K5088677] in T. officinale
Figure 14: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.
Cycle 5 - An Improved Design
The insights from cycle 4 led us to conclude that further optimization of our ratiometric test construct was necessary. To address this, we designed an enhanced version capable of characterizing a broader range of regulatory elements. Based on the results of our 3'UTR characterization, we replaced the 35S 3'UTR with the strongest one we identified (T_pgm, Phosphoglucomutase, [BBa_K5088109]) to boost expression levels. Additionally, we optimized the constructs by incorporating brighter reporter genes, switching from eGFP to StayGold and from mCherry to mScarlet-I3. These upgrades allow for more precise and sensitive measurements, advancing our ability to characterize regulatory elements more effectively across diverse applications.
Furthermore, we are planning to implement additional optimization steps for our protoplast and leaf infiltration protocols to improve the efficiency and reliability of our transient expression systems. These enhancements are crucial for obtaining consistent results with the remaining promoter + 5'UTR parts. By refining both our constructs and testing methods, we are moving closer to developing robust tools for dandelion genetic engineering.
For more details on these improvements, please refer to our plant transformation section.
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
S. Amack, M. Antunes. CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. Crossref
O. Shimomura, F. Johnson, Y. Saiga. Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. Crossref
D. Jacob, A. Lewin, B. Meister, B. Appel. Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery. Crossref
J. Brophy, K. Magallon, L. Duan, V. Zhong, P. Ramachandran, K. Kniazev, J. Dinneny. Synthetic genetic circuits as a means of reprogramming plant roots. Crossref
K. Schaumberg, M. Antunes, T. Kassaw, W. Xu, C. Zalewski, J. Medford, A. Prasad. Quantitative characterization of genetic parts and circuits for plant synthetic biology. Crossref
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