Part:BBa_J428074
Plant promoters CaMV35S
35S is a plant specific promoter obtained from the Cauliflower Mosaic Virus. The part is intended for use as a constitutive promoter for gene expression experiments in plants. This part is compatible with GoldenGate MoClo Assembly Standard as it is free from internal BsaI and BpiI recognition sequences.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 226
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution by iGEM Marburg 2024
In the early stages of our project we noticed a severe lack of genetic tools for the engineering of Taraxacum kok-saghyz (TKS) and Taraxacum officinale (TO). Interviews with Prof. Dr. Dirk Prüfer and Dr. Fred Eickmeyer underscored that this lack is one of the major bottlenecks hampering ambitious engineering efforts which would pave the way for dandelion to become a crop of the future.
In our discussions with several experts we talked about changes to European Union (EU) regulations regarding genetically modified organisms (GMOs). The EU Parliament is considering exempting certain plants developed through New Genomic Techniques (NGTs) from existing GMO laws. To align with these potential regulatory shifts and ensure that these engineered plants could be used within the EU, we chose to focus on developing endogenous regulatory elements.
However, to set these ambitious plans into motion, we first needed a suitable starting ground. For this, we performed extensive literature research, compiling a list of various regulatory elements which have previously been used. One of the parts which stood out for its extensive use was the Cauliflower Mosaic virus 35S promoter (1 ,2, 3 ,4 ,5 ,6 ), which shows ubiquitous gene expression across a wide range of plant species(7). Lucky for us, this exact promoter was included in the iGEM distribution kit under the registry page: BBa_J428074.
Even though the CaMV35S promoter is one of the most used parts in Plant Synthetic Biology, we were surprised to find that not much information was contained on the corresponding iGEM registry page. In order to document our efforts and help future iGEM teams with useful information, we have added much needed information to this page.
In our project we were heavily relying on the 35S promoter and in the following we describe how we used it to be able to test genetic regulatory elements in both Nicotiana benthamiana, Taraxacum kok-saghyz and Taraxacum officinale.
In the beginning of our project, we created a plasmid termed Tarakate - Test construct RUBY [BBa_K5088676] which includes the RUBY reporter(8) 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, 2, 3) using the leaf infiltration method established by the iGEM Team Bielefeld 2021, generated transient RUBY expression in TO and TKS using leaf infiltration (Figure 4) as well as in TKS using the cut-dip budding method(9) (Figure 5).
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 (10).
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:Expression of the RUBY reporter in Taraxacum kok-saghyz using cut-dip-budding transformation. (A) T. kok-saghyz bud after transformation. (B) Close-up of the transformed bud showing pigmented tissues, indicating RUBY expression. (C) Development of roots with distinctive red pigmentation characteristic of RUBY expression. (D) Absorbance spectrum showing a clear peak around 535 nm in the transformed line (TKS_pRUBY_CDB) compared to the wild type (TKS_WT), demonstrating successful integration and expression of the RUBY reporter.
Next, we used the exact same architecture as the RUBY plasmid, but exchanged the coding sequence for GFP. This plasmid was the next step into the direction of quantitative characterization of endogenous regulatory parts of TKS. This plasmid was named Tarakate - Test construct GFP [BBa_K5088675] and tested in N. benthamiana. Our results show a clear difference between WT plants treated with A. rhizogenes K599 and A. tumefaciens GV3101 P19 [negative control] (Figure 6 A, B) and plants infiltrated with the GFP plasmid (Figure 6 C, D).
Figure 6: WT Nb plants treated with A. rhizogenes K599 and A. tumfaciens GV3101 P19 (negative control) under (A) normal light conditions and (B) under long-wave UV light (395nm). Nb infiltrated with Tarakate - Test construct GFP under (C) normal light conditions and (D) under long-wave UV light (395nm).
Figure 7: Boxplot showing successful GFP expression in N. benthamiana. Data was generated from individual leaf disks placed abaxial side down into 96 black well plates with clear bottom. 5µl of water was placed in the wells in order for the disks to adhere to the plate. Expression was measured from the bottom using the following parameters: Excitation: 408nm ±6nm; Emission: 510nm ±6nm (average from matrix scan).
For our part characterization, we built the plasmid: Tarakate - Consensus measurement construct [BBa_K5088677] (Figure 8). This plasmid consists of two genes for ratiometric characterization of genetic regulatory elements. The first cassette uses the exact same regulatory elements as the previous two plasmids and is used to integrate endogenous parts of TKS in either the Promoter + 5’UTR or the 3’UTR position. The mCherry cassette is used to normalize the data in order to reduce variability and increasing consistency between experiments. The Consensus measurement construct was tested in N. benthamiana and showed great signal-to-noise ratio for both fluorescent reporters (Figure 9, 10 and 11).
Figure 8: SBOL scheme of Tarakate - Consensus measurement construct [BBa_K5088677].
Figure 9: WT Nb plants treated with A. rhizogenes K599 and A. tumfaciens GV3101 P19 (negative control) under (A) normal light conditions and (B) under long-wave UV light (395nm). Nb infiltrated with Tarakate - Consensus measurement construct [BBa_K5088677] under (C) normal light conditions and (D) under long-wave UV light (395nm). [Note: In our ratiometric measurement construct we opted to use eGFP over avGFP which was used in the other GFP construct; avGFP favors UV light and therefore the signal under long-wave UV is brighter compared to here; read more about our reporters here]
Figure 10: Boxplot showing successful mCherry expression from the Tarakate - Consensus measurement construct in N. benthamiana. Data was generated from individual leaf disks placed abaxial side down into 96 black well plates with clear bottom. 5µl of water was placed in the wells in order for the disks to adhere to the plate. Expression was measured from the bottom using the following parameters: Excitation: 470nm ±6nm; Emission: 507nm ±6nm (average from matrix scan).
Figure 11: Boxplot showing successful mCherry expression from the Tarakate - Consensus measurement construct in N. benthamiana. Data was generated from individual leaf disks placed abaxial side down into 96 black well plates with clear bottom. 5µl of water was placed in the wells in order for the disks to adhere to the plate. Expression was measured from the bottom using the following parameters: Excitation: 570nm ±6nm; Emission: 610±6nm (average from matrix scan).
References
[1] Collins-Silva, J., Nural, A. T., Skaggs, A., Scott, D., Hathwaik, U., Woolsey, R., Schegg, K., McMahan, C., Whalen, M., Cornish, K., & Shintani, D. (2012). Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism. In Phytochemistry (Vol. 79, pp. 46–56). Elsevier BV. https://doi.org/10.1016/j.phytochem.2012.04.015
[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] Stolze, A., Wanke, A., van Deenen, N., Geyer, R., Prüfer, D., & Schulze Gronover, C. (2017). Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway. In Plant Biotechnology Journal (Vol. 15, Issue 6, pp. 740–753). Wiley. https://doi.org/10.1111/pbi.12672
[4] van Deenen, N., Unland, K., Prüfer, D., & Schulze Gronover, C. (2019). Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber. In Molecules (Vol. 24, Issue 15, p. 2703). MDPI AG. https://doi.org/10.3390/molecules24152703
[5] Wolters, S. M., Benninghaus, V. A., Roelfs, K.-U., van Deenen, N., Twyman, R. M., Prüfer, D., & Schulze Gronover, C. (2023). 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. In Frontiers in Plant Science (Vol. 14). Frontiers Media SA. https://doi.org/10.3389/fpls.2023.1228961
[6] Benninghaus, V. A., van Deenen, N., Müller, B., Roelfs, K.-U., Lassowskat, I., Finkemeier, I., Prüfer, D., & Schulze Gronover, C. (2019). Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology. In B. Usadel (Ed.), Journal of Experimental Botany (Vol. 71, Issue 4, pp. 1278–1293). Oxford University Press (OUP). https://doi.org/10.1093/jxb/erz512
[7] Amack, S. C., & Antunes, M. S. (2020). CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. In Current Plant Biology (Vol. 24, p. 100179). Elsevier BV. https://doi.org/10.1016/j.cpb.2020.100179
[8] 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
[9] 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
[10] 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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