Part:BBa_K5088676
Tarakate - Test construct RUBY
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 230
Illegal BglII site found at 4240
Illegal BamHI site found at 2889
Illegal XhoI site found at 4485 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 825
Illegal NgoMIV site found at 1404
Illegal NgoMIV site found at 3117 - 1000COMPATIBLE WITH RFC[1000]
Background
In the beginning of our project, we created a plasmid termed Tarakate - Test construct RUBY [BBa_K5088676] which includes the RUBY reporter(1) previously introduced to the iGEM competition by the iGEM team Bielefeld 2021. RUBY is a reporter that ultimately results in the biosynthesis of betalain, a pigment which results in red coloration of plant tissue. We chose this reporter, because successful expression is directly visible by eye and does not require any additional equipment for the measurement of the reporter. The coding sequence is driven by the 35S promoter, the 5’UTR region of the Tobacco Mosaic Virus (TMV), called omega sequence and the 35S 3’UTR. We used this plasmid extensively in the beginning of our project in order to familiarize ourselves, optimize and troubleshoot different transformation protocols and were able to generate RUBY expression in Nicotiana benthamiana (Figure 1-3) using the leaf infiltration method established by the iGEM Team Bielefeld 2021, generated transient RUBY expression in Taraxacum officinale (TO) and Taraxacum kok-saghyz (TKS) using leaf infiltration (Figure 4) as well as in TKS using the cut-dip budding method(2) (Figure 5).
Results
Figure 1: (A)Nicotiana benthamiana plants infiltrated with A. rhizogenes K599 and A. tumefaciens GV3101 P19 (negative control). B: N. benthamiana plants treated with A. rhizogenes K599 harboring Tarakate - Test construct RUBY [BBa_K5088676] and A. tumefaciens GV3101 P19. The transgenic plants exhibit a strong pigmentation compared to the WT due to the production of betalains. Images were taken 3 days after infiltration (dai).
Figure 2: Absorbance spectrum [450nm-600nm] of N. benthamiana infiltrated plants with Tarakate - Test construct RUBY [BBa_K5088676]. Shown are two curves from two individual plants. Plotted is the mean absorbance values of 2 technical replicates of 2 different infiltration spots of a single leaf of one plant. The protocol for Betalain extraction was taken from iGEM Bielefeld 2021.
Figure 3: Boxplot showing the absorbance level at 535nm of transgenic N. benthamiana plants and non-treated plants. This measurement was done in 96 well plate format in clear plates. Each point corresponds to a single infiltration spot which was measured by two technical replicates. Protocol was taken from Chiang et al 2024 (3).
Figure 4: (A) WT TKS showing no RUBY expression (B) Successful RUBY expression in TKS (C) Two leaf disks from samples of (B) were excised and betalain was extracted according to the method introduced by iGEM Bielefeld 2021. The absorbance spectrum was measured in a clear 96 well plate using a platereader.
Figure 5: Absorbance spectrum of stably transformed Taraxacum kok-saghyz (TKS) shoot in comparison to WT TKS. The curve from the transgenic roots is clearly distinguishable from the WT sample.
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
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References
[1] He, Y., Zhang, T., Sun, H., Zhan, H., & Zhao, Y. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. In Horticulture Research (Vol. 7, Issue 1). Oxford University Press (OUP). https://doi.org/10.1038/s41438-020-00390-1
[2] Cao, X., Xie, H., Song, M., Lu, J., Ma, P., Huang, B., Wang, M., Tian, Y., Chen, F., Peng, J., Lang, Z., Li, G., & Zhu, J.-K. (2023). Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture. In The Innovation (Vol. 4, Issue 1, p. 100345). Elsevier BV. https://doi.org/10.1016/j.xinn.2022.100345
[3] Chiang, B., Lin, K., Chen, Y., Huang, C., Goh, F., Huang, L., Chen, L., & Wu, C. (2024). Development of a tightly regulated copper‐inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase. In New Phytologist (Vol. 244, Issue 1, pp. 318–331). Wiley. https://doi.org/10.1111/nph.20021
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