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With this in mind, we set out to evaluate the current repertoire of regulatory parts used in <i>T. kok-saghyz</i> (TKS). Our review revealed that most constructs developed for TKS rely heavily on a limited selection of regulatory elements, such as the Cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (NOS) terminator.
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<caption><b>Table 1:</b> Regulatory elements identified in T. kok-saghyz literature.</caption>
 
<caption><b>Table 1:</b> Regulatory elements identified in T. kok-saghyz literature.</caption>
 
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With this in mind, we set out to evaluate the current repertoire of regulatory parts used in <i>T. kok-saghyz</i> (TKS). Our review revealed that most constructs developed for TKS rely heavily on a limited selection of regulatory elements, such as the Cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (NOS) terminator.
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To overcome this challenge, our project this year focused on developing a broader collection of regulatory parts specifically tailored for TKS. By increasing the diversity of genetic components, we aim to support more ambitious synthetic biology projects in this promising plant.
 
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====Political Context====
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====Political & Regulatory Aspect====
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To ensure that engineered plants can be utilized, it is crucial that they adhere to the regulatory standards of the countries in which they will be deployed. In the European Union (EU), regulations surrounding genetically modified organisms (GMOs) are particularly strict and have sparked intense debate. However, a relaxation of these stringent regulations may be on the horizon.<br>
 +
The EU Parliament recently adopted its position for negotiations with member states regarding the proposal on New Genomic Techniques (NGTs). Currently, all plants developed using NGTs are regulated under the same framework as GMOs. However, Members of the European Parliament support the creation of two distinct categories with corresponding regulatory frameworks for NGT plants. NGT plants deemed equivalent to conventionally bred plants (NGT 1) would be exempt from the GMO legislation, while NGT plants that do not meet this criterion (NGT 2) would still be subject to more stringent regulations.
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If this proposal is enacted into European law, engineered plants intended for use within the EU must meet the criteria for NGT 1 status. To achieve this, it is crucial to prioritize the use of endogenous regulatory elements over synthetic ones to fulfill the strict requirements outlined in the proposal.
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===Plant Synthetic Biology===
 
===Plant Synthetic Biology===
 
====Gene Structure in Plants====
 
====Gene Structure in Plants====
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Gene structure in plants, like in other eukaryotes, is characterized by a complex organization of coding and non-coding regions. Unlike the typically continuous coding sequences found in prokaryotes, plant genes consist of exons—both protein-coding and non-coding—separated by introns, which are removed during RNA splicing to produce mature mRNA.
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Untranslated regions (UTRs) at both ends of the mRNA also play crucial roles. The 5' UTR influences translation initiation, while the 3' UTR affects mRNA stability and lifespan. These regions enable plants to finely tune protein production in response to environmental and developmental cues.
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Promoters, located upstream of the coding sequence, are critical for initiating transcription. These regions contain specific sequences, that regulate when, where, and how much a gene is expressed. The terminator region downstream ensures proper transcription termination and mRNA processing.
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In contrast to prokaryotes, where genes are often organized in operons and transcribed as polycistronic mRNA, plant genes are transcribed independently. This complexity, including the roles of introns, UTRs, and promoters, is essential for regulating gene activity in processes like development, differentiation, and environmental response.
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<figcaption><p><b>Figure 2:</b> Schematic representation of gene expression in plants, illustrating the process from transcription to translation. RNA Polymerase II transcribes the DNA into a primary RNA transcript, which then undergoes processing, including 5' capping, 3' end processing, and polyadenylation. The pre-mRNA is spliced to remove introns and produce mature mRNA, which is exported from the nucleus to the cytoplasm. In the cytoplasm, the mRNA is translated into protein and directed to its subcellular location.
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====Cis-Regulatory Elements====
 
====Cis-Regulatory Elements====
  

Revision as of 15:53, 27 September 2024


Protein tyrosine kinase - Promoter+5'UTR from T. kok-saghyz


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.


Lack of Endogenous Regulatory Parts

To successfully implement these engineering efforts, it is essential to have a set of well-characterized regulatory parts, including promoters, 5’ untranslated regions (UTRs), 3’ UTRs, and other genetic sequences that provide precise control over gene expression. These standardized components are fundamental for achieving reliable and predictable outcomes in any synthetic biology project.

With this in mind, we set out to evaluate the current repertoire of regulatory parts used in T. kok-saghyz (TKS). Our review revealed that most constructs developed for TKS rely heavily on a limited selection of regulatory elements, such as the Cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (NOS) terminator.

Part Type Part Name Origin References
Promoter + 5’UTR 35S Cauliflower mosaic virus (9, 10, 11, 12, 13, 14)
Promoter + 5’UTR NOS Agrobacterium tumefaciens (9)
Promoter + 5’UTR PEP16 Hevea brasiliensis (15)
Promoter + 5’UTR Ubiquitin4-2 Petroselinum crispum (16)
Promoter UBQ1 Arabidopsis thaliana (10)
Promoter U6-26 Arabidopsis thaliana (10)
Promoter CPTL1 Taraxacum kok-saghyz (17)
3'UTR 35S Cauliflower mosaic virus (9, 10, 11, 12, 13, 14)
3'UTR NOS Agrobacterium tumefaciens (9)
3'UTR UBQ1 Arabidopssi thaliana (10)
3'UTR 3A Pisum sativum (16)
Table 1: Regulatory elements identified in T. kok-saghyz literature.

To overcome this challenge, our project this year focused on developing a broader collection of regulatory parts specifically tailored for TKS. By increasing the diversity of genetic components, we aim to support more ambitious synthetic biology projects in this promising plant.

Political & Regulatory Aspect

To ensure that engineered plants can be utilized, it is crucial that they adhere to the regulatory standards of the countries in which they will be deployed. In the European Union (EU), regulations surrounding genetically modified organisms (GMOs) are particularly strict and have sparked intense debate. However, a relaxation of these stringent regulations may be on the horizon.
The EU Parliament recently adopted its position for negotiations with member states regarding the proposal on New Genomic Techniques (NGTs). Currently, all plants developed using NGTs are regulated under the same framework as GMOs. However, Members of the European Parliament support the creation of two distinct categories with corresponding regulatory frameworks for NGT plants. NGT plants deemed equivalent to conventionally bred plants (NGT 1) would be exempt from the GMO legislation, while NGT plants that do not meet this criterion (NGT 2) would still be subject to more stringent regulations.

If this proposal is enacted into European law, engineered plants intended for use within the EU must meet the criteria for NGT 1 status. To achieve this, it is crucial to prioritize the use of endogenous regulatory elements over synthetic ones to fulfill the strict requirements outlined in the proposal.

Plant Synthetic Biology

Gene Structure in Plants

Gene structure in plants, like in other eukaryotes, is characterized by a complex organization of coding and non-coding regions. Unlike the typically continuous coding sequences found in prokaryotes, plant genes consist of exons—both protein-coding and non-coding—separated by introns, which are removed during RNA splicing to produce mature mRNA.

Untranslated regions (UTRs) at both ends of the mRNA also play crucial roles. The 5' UTR influences translation initiation, while the 3' UTR affects mRNA stability and lifespan. These regions enable plants to finely tune protein production in response to environmental and developmental cues.

Promoters, located upstream of the coding sequence, are critical for initiating transcription. These regions contain specific sequences, that regulate when, where, and how much a gene is expressed. The terminator region downstream ensures proper transcription termination and mRNA processing.

In contrast to prokaryotes, where genes are often organized in operons and transcribed as polycistronic mRNA, plant genes are transcribed independently. This complexity, including the roles of introns, UTRs, and promoters, is essential for regulating gene activity in processes like development, differentiation, and environmental response.

Figure 2: Schematic representation of gene expression in plants, illustrating the process from transcription to translation. RNA Polymerase II transcribes the DNA into a primary RNA transcript, which then undergoes processing, including 5' capping, 3' end processing, and polyadenylation. The pre-mRNA is spliced to remove introns and produce mature mRNA, which is exported from the nucleus to the cytoplasm. In the cytoplasm, the mRNA is translated into protein and directed to its subcellular location.


Cis-Regulatory Elements

Design and Characterization

Identification & Design Strategy

Genetic Context of the Part

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GO ribbon for PTI1

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Measurement

Dual Fluorescence Reporter Assay

Choice of Reporter Genes

Transient Transformation

Leaf Infiltration

Protoplast

Measurement Setup

Results

The Dandelion Toolbox

Overview

Part Identifier Part Type Nickname Part Description
BBa_K5088000 Promoter + 5'UTR P_PTI1 Protein tyrosine kinase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088001 Promoter + 5'UTR P_RPL28 Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088002 Promoter + 5'UTR P_GSK3B Glycogen synthase kinase 3 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088003 Promoter + 5'UTR P_MGRN1 E3 ubiquitin-protein ligase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088004 Promoter + 5'UTR P_betB Betaine-aldehyde dehydrogenase - Promoter+5'UTR from T. kok-saghyz
BBa_K5088005 Promoter + 5'UTR P_pgm Phosphoglucomutase - 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_K5088009 Promoter + 5'UTR P_CUL1 Cullin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088010 Promoter + 5'UTR P_VPS4 Vacuolar protein-sorting-associated protein 4 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088011 Promoter + 5'UTR P_EIF2S3 Translation initiation factor 2 subunit 3 - 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_K5088050 Inducible Promoter + 5'UTR P_HSP12.6 HSP12.6 - Heat inducible promoter+5'UTR from T. koksaghyz
BBa_K5088051 Inducible Promoter + 5'UTR P_HSP23.5 HSP23.5 - Heat inducible promoter+5'UTR from T. koksaghyz
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: Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.

Dandelion Handbook

References

[1] J. B. van Beilen, Y. Poirier, Establishment of new crops for the production of natural rubber. Trends Biotechnol. 25, 522–529 (2007).

[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).

[7] D. A. Ramirez-Cadavid, K. Cornish, F. C. Michel, Taraxacum kok-saghyz (TK): compositional analysis of a feedstock for natural rubber and other bioproducts. Ind. Crops Prod. 107, 624–640 (2017).

[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).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1262
    Illegal XbaI site found at 184
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1262
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1262
    Illegal BglII site found at 409
    Illegal XhoI site found at 436
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1262
    Illegal XbaI site found at 184
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1262
    Illegal XbaI site found at 184
    Illegal AgeI site found at 1761
  • 1000
    COMPATIBLE WITH RFC[1000]