Regulatory
pTriple Op

Part:BBa_K4213003

Designed by: Ioannis Retalis   Group: iGEM22_Thessaly   (2022-08-26)


modified CaMV p35S to be repressed by TetR

Overview

The Triple Op Promoter (or pTriple Op) is a modified version of the constitutive promoter 35S from the plant pathogen Cauliflower Mosaic Virus (CaMV). The CaMV 35S promoter (p35S) is arguably the most well-studied and experimentally used regulatory component with activity in plant cells [1]. With abundant information available on its individual domains, its known modular architecture allows for the development of both inducible and repressible gene expression. This meant that we could incorporate Tet Operator (TetO) sequences within the promoter sequence, placing the second construct under the influence of TetR. Therefore, we introduced three repeats of TetO to p35S and set them in close proximity to the TATA box, mimicking the design of others before us [2], as this is currently considered the best way to achieve maximum repression with TetR in planta. To confirm its functionality, we designed a Reporter System composed of the basic parts tetO7:pTripleOp:mVenus:tNOS (BBa_K4213041).

Experimental Design and Results

We selected Ν. benthamiana since it allows for high and rapid expression of transgenes, by agroinfiltration, as the species is quite susceptible to plant viral vectors [3]. N. benthamiana leaf samples are also able to be observed under UV lumination for expression of fluorescent proteins [3]. As for the cloning method, we opted for the Golden Braid for its simplicity, effectiveness and rapidness [4] and managed to confirm assembly with the help of diagnostic digestion.

Diagnostic_Digestion_of_TetO7_pTriple_Venus_tNOS(s)
Figure 1: Diagnostic Digestion of “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387.

Following that, came agroinfiltration and observation under the Plate Reader, 4 days post infiltration (dpi) [5], whereupon we confirmed the functionality of the Transcriptional Unit (TU).

mVenus_fluorescence_under_pTriple
Figure 2: Fluorescence of different leaf samples measured under the Plate Reader.

References

[1] Amack, S. C., & Antunes, M. S. (2020b, December). CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. Current Plant Biology, 24, 100179. https://doi.org/10.1016/j.cpb.2020.100179

[2] Gatz, C., Frohberg, C., & Wendenburg, R. (1992d, May). Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. The Plant Journal, 2(3), 397–404. https://doi.org/10.1046/j.1365-313x.1992.t01-37-00999.x

[3] Pombo, M. A., Rosli, H. G., Fernandez-Pozo, N., & Bombarely, A. (2020d). Nicotiana benthamiana, A Popular Model for Genome Evolution and Plant–Pathogen Interactions. The Tobacco Plant Genome, 231–247. https://doi.org/10.1007/978-3-030-29493-9_14

[4] Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013d, May 13). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618–1631. https://doi.org/10.1104/pp.113.217661

[5] Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008d, August). Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions. Molecular Plant-Microbe Interactions®, 21(8), 1015–1026. https://doi.org/10.1094/mpmi-21-8-1015


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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