Part:BBa_K4213029
pNOS:Venus:tNOS
Engineering Cycle 2: First Iteration: Deciding on Regulatory elements
Design
The Reporter System
Starting with the promoter of choice, we opted for the Nopaline Synthase promoter (NOS promoter) as it is a promoter proven to work in plants [1] and a standardized Phytobrick [2] that is weaker than the obvious counterpart (CaMV 35s promoter) [3]. Lower expression meant less possibility for toxicity symptoms caused by high expression of TetR [4] and for better responsiveness of the system to tetracycline. Continuing with the reporter system, mVenus [5] was the protein of choice as this is an autofluorescent protein (AFP) (Excitation λ: 515 nm, Emission λ: 527 nm,[6]) that is well characterized in Nicotiana benthamiana. We paid careful attention to the Kozak frame of the protein and given that, in green plants, a characteristic motif has been observed (R(-3) M(-2) –(+1) A(+1) U(+2) G(+3) G(+4) C(+5)) [7], we made two substitutions (4:T>C, 5:G>T) to the ORF leading to a mutation (V2A) in the amino acid (aa) sequence, which should not cause folding and activity mishaps due to the position and size of the two aa. Lastly, what remains is the terminator. Settling, again, on Nopaline Synthase (NOS terminator), this is a strong terminator that is a standardized Golden Braid part [8]. Putting it all together, we created a promising Reporter System, consisting of pNOS:mVenus:tNOS.
Experimental Design-Plant Selection
While our end-product was already decided to be the plant P. australis, we needed a stepping stone, a model plant to perform the first characterization and optimization of our constructs. For this, 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 [9]. N. benthamiana leaf samples are also able to be observed under UV lumination for expression of fluorescent proteins [9].
Experimental Design-Cloning method
Among the prevalent cloning methods used today, one that stands behind its simplicity, effectiveness and rapidness is the Golden Braid method [10]. Based on the Golden Gate system, this technology relies on the Type IIS restriction enzymes to alternate between level Alpha and Omega states, resulting in the assembly of one or more Transcriptional Units (TUs) One of the main benefits of Golden Braid is the four nucleotide cutting sequence of the restriction enzymes, which can be completely random, equipping it with the ability to completely fix the order of insertion between different parts by designing different complementary four-nucleotide overhangs at each end. For our assembly, we used the proposed sequences of the technique.
Build
Next came the cloning experiments that lead to the assembly of our Reporter System. The procedure was conducted on a special strain of E. coli, K-12 DH5a, engineered to maximize transformation efficiency. With the help of diagnostic digestion and analysis by gel electrophoresis we were able to confirm the assembly of our construct.
Test
As mentioned above, we selected N. benthamiana for our experiments, so what followed was the transient expression of our constructs in planta through agroinfiltration. This method allows for noticeable expression of transgenes within 4 days of infiltration [11] and, with the fluorescent mVenus, we planned to observe transgene expression under UV luminescence using fluorescent microscopy.
Learn
After examining our samples under the confocal microscope and taking our pictures we concluded that our transcriptional unit did manage to express mVenus in great numbers and, therefore, we moved on to the second iteration of this DBTL cycle, to test our design with the repressor.
References
[1] González-Grandío, E., Demirer, G. S., Ma, W., Brady, S., & Landry, M. P. (2021b, September 14). A Ratiometric Dual Color Luciferase Reporter for Fast Characterization of Transcriptional Regulatory Elements in Plants. ACS Synthetic Biology, 10(10), 2763–2766. https://doi.org/10.1021/acssynbio.1c00248
[2] Addgene: pUPD+pNOS. (n.d.-c). Retrieved October 9, 2022, from https://www.addgene.org/170875/
[3] Sanders, P., Winter, J., Barnason, A., Rogers, S., & Fraley, R. (1987c). 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] CORLETT, J. E., MYATT, S. C., & THOMPSON, A. J. (1996b, April). Toxicity symptoms caused by high expression of Tet represser in tomato (Lycopersicon esculentum Mill. L.) are alleviated by tetracycline. Plant, Cell and Environment, 19(4), 447–454. https://doi.org/10.1111/j.1365-3040.1996.tb00336.x
[5] Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002b, January). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology, 20(1), 87–90. https://doi.org/10.1038/nbt0102-87
[6] Lambert, T. (n.d.-b). mVenus at. FPbase. Retrieved October 9, 2022, from https://www.fpbase.org/protein/mvenus/
[7] Hernández, G., Osnaya, V. G., & Pérez-Martínez, X. (2019b, December). Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes. Trends in Biochemical Sciences, 44(12), 1009–1021. https://doi.org/10.1016/j.tibs.2019.07.001
[8] Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013e, 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
[9] Pombo, M. A., Rosli, H. G., Fernandez-Pozo, N., & Bombarely, A. (2020e). 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
[10] Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013f, 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
[11] Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008e, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 192
Illegal NheI site found at 297 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1013
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |