Part:BBa_K4213018
TetR:NLS
Overview
Repressors have been widely used in synthetic biology as they allow for a precise control of gene expression, producing a desired phenotype. Amid the field, are repressors that can function properly in plants and amongst the most prevalent ones are the Tet Repressors [1]. Tet Repressor proteins (or TetRs) are proteins that play a vital role in antibiotic resistance against Tetracycline. By looking into the repressor’s role in Tetracycline-induced transcriptional control, it was understood that the suppression occurred only in the adjacent presence of specific DNA sequences called Tet Operators (or TetOs) [2]. And so, there were two problems that needed solving; which TetR to choose and how to place the TetO sequences. The registry of biological parts already provided standardized parts for the two of them (BBa_R0040, BBa_C0040), so what remained were a few tweaks in order to achieve proper expression in a plant system.
Tet Repressor for plants
Regarding the first one and having learnt that the reporter syntax works as intended (BBa_K4213029), it was time to substitute the fluorescent protein with our repressor of choice. As already mentioned, the registry gave as the base sequence for TetR (BBa_C0040), but this part was intended for expression in prokaryotic organisms and not eukaryotic ones. For that reason, we needed to anticipate the presence of a nucleus, possible splicing sites and unoptimized expression on account of less frequently used synonymous codons present in the coding sequence (cds). Starting with the first, it seemed that due to the size of TetR (48 kDa) diffusion into the cell nucleus was more than likely [3]. Nevertheless, we decided to add a Nuclear Localization Signal (NLS), specifically Simian Virus 40 NLS [4], to the sequence since this would ensure its passage into the nucleus. Moving on to the possible splicing sites, the now NLS-containing sequence was run through a neural network based program called NetGene2. This method is proven to predict possible splicing sites within given sequences, based on analysis made on the plant Arabidopsis thaliana [5]. Analysis showed no potential splicing sites within the cds so we moved on with no modifications. Concluding with codon optimization, this is a tool revolving around the frequency that each synonymous codon occurs within a certain organism’s genome. We used IDT’s codon optimization tool for expression in Nicotiana benthamiana.
It is worth pointing out that, for the visualization of expression, we, again, decided to incorporate the modified mVenus into the sequence, leading to a construct consisting of pNOS:Venus:TetR:NLS:tNOS (BBa_K4213030).
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 [6]. N. benthamiana leaf samples are also able to be observed under UV lumination for expression of fluorescent proteins [6]. As for the cloning method, we opted for the Golden Braid for its simplicity, effectiveness and rapidness [7] and managed to confirm assembly with the help of diagnostic digestion.
Following that, came agroinfiltration and observation under the confocal microscope 4 days post infiltration (dpi) [8], whereupon we confirmed the functionality of the Transcriptional Unit (TU).
References
[1] Andres, J., Blomeier, T., & Zurbriggen, M. D. (2019c, January 28). Synthetic Switches and Regulatory Circuits in Plants. Plant Physiology, 179(3), 862–884. https://doi.org/10.1104/pp.18.01362
[2] Ramos, J. L., Martínez-Bueno, M., Molina-Henares, A. J., Terán, W., Watanabe, K., Zhang, X., Gallegos, M. T., Brennan, R., & Tobes, R. (2005b, June). The TetR Family of Transcriptional Repressors. Microbiology and Molecular Biology Reviews, 69(2), 326–356. https://doi.org/10.1128/mmbr.69.2.326-356.2005
[3] Gatz, C., & Quail, P. H. (1988d, March). Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proceedings of the National Academy of Sciences, 85(5), 1394–1397. https://doi.org/10.1073/pnas.85.5.1394
[4] Lu, J., Wu, T., Zhang, B., Liu, S., Song, W., Qiao, J., & Ruan, H. (2021d, May 22). Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Communication and Signaling, 19(1). https://doi.org/10.1186/s12964-021-00741-y
[5] Hebsgaard, S. (1996b, September 1). Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Research, 24(17), 3439–3452. https://doi.org/10.1093/nar/24.17.3439
[6] Pombo, M. A., Rosli, H. G., Fernandez-Pozo, N., & Bombarely, A. (2020c). 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
[7] Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013c, 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
[8] Goodin, M. M., Zaitlin, D., Naidu, R. A., & Lommel, S. A. (2008c, 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]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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