Part:BBa_K4213023

NOS promoter without Kozak frame

Overview

The Nopaline Synthase Promoter (or NOS Promoter) is a regulatory element of bacterial origin that can drive expression in plant tissues. Normally, it is used for generation of an unusual compound, called Nopaline, that, when secreted into the environment, can be used by Agrobacterium tumefaciens as a nutrient source ^[1]. In synthetic biology, wherein transgene expression can be achieved through the creation of novel devices, pNOS has been established as a Phytobrick for the ability to constitutively express genes in Plant Tissues ^[2]. In comparison to the obvious counterpart, the Cauliflower Mosaic Virus 35s Promoter (CaMV 35s Promoter), it leads to lower levels of expression ^[3], desirable in conditions where increased transgene presence results in toxicity to the host or a non-responsive device. iGEM Thessaly 2022 has managed to confirm the functionality of this part by designing a Reporter System composed of the basic parts pNOS:mVenus:tNOS (BBa_K4213029).

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 ^[4]. N. benthamiana leaf samples are also able to be observed under UV lumination for expression of fluorescent proteins ^[4]. As for the cloning method, we opted for the Golden Braid for its simplicity, effectiveness and rapidness ^[5] and managed to confirm assembly with the help of diagnostic digestion.

Figure 1: Diagnostic Digestion of pDGB3α2_pNOS-Venus-tNOS with HindIII and expected bands (bp): 6345, 1033 and 509.

Following that, came agroinfiltration and observation under the confocal microscope 4 days post infiltration (dpi) ^[6], whereupon we confirmed the functionality of the Transcriptional Unit (TU).

Figure 2: Agroinfiltration experiment: positive (pDGB3α2_pNOS-Venus-tNOS) control. The red arrows show intercellular bridges that confirm that Venus protein is in the cytoplasm.

References

[1] Kim, Y., An, G., (1997) Characterization of a hexamer motif and b element of the nopaline (nos) promoter. Molecules and Cells. PMID: 9085270

[2] Addgene: pUPD+pNOS. (n.d.-b). Retrieved October 9, 2022, from https://www.addgene.org/170875/

[3] Sanders, P., Winter, J., Barnason, A., Rogers, S., & Fraley, R. (1987b). Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucleic Acids Research, 15(4), 1543–1558. https://doi.org/10.1093/nar/15.4.1543

[4] Pombo, M. A., Rosli, H. G., Fernandez-Pozo, N., & Bombarely, A. (2020b). 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

[5] Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013b, 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

[6] Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008b, 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 188
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]