Part:BBa_K4213019
TetR:NLS:KRAB
Inspiration
‘Leaky’ gene expression, wherein expression of a regulated gene is observed in its uninduced state, is a rather persistent problem in synthetic biology. Whereas, low levels of leaky expression can be considered a feature in nature, required for the inducible expression of a toxic gene, here is more of a remaining unknown variable to untested genetic circuits, leading to poor performance. Ligand-inducible genetic control systems, like our Repressor module, lie in the bedrock of synthetic biology and are no strangers to this problem. The Tetracycline Repressor (TetR), originally meant for bacterial based inducible expression platforms, has been introduced to plant synthetic systems for quite some time now and, without exception, shows, or rather, exaggerates the issue.
Our Part of Choice
Searching through the Registry, we stumbled upon a Part (BBa_C0040) that contained the coding sequence for TetR. Being introduced as a standard biological part from the early days of iGEM, the level of characterization was impressive, containing information about dynamic range, reversibility of function and even toxicity to its host. Absent, though, was this level of characterization in planta. Having a closer look at the part sequence, present, apart from the TetR, was an LVA tail for a faster fall time, degradation, of the protein [1] and absent was a Ribosome Binding Site (RBS) needed for bacterial expression. It was evident that, for the system to be optimized for plant expression, modifications needed to be made.
Design of the Improved Part
We decided to use a part fitter for eukaryotic systems, the TetR-KRAB transregulator. As the name suggests, TetR has been fused with the Krüppel Associated Box protein (KRAB) [2], enhancing the inhibition abilities of the complex by adding another mechanism of silencing [3]. This, along with the proper tetracycline Operator engineering for which you can learn about in the Engineering page of our wiki, would ensure minimum leakiness of the system. In regards to the rest of the sequence, we decided to remove the LVA tail and substitute it with a Nuclear Localization Signal (NLS). Even though, due to the size of TetR, diffusion into the cell nucleus seemed more than likely [4], the addition of the Simian Virus 40 NLS [5] would ensure passage into the cell’s nucleus. Some final modifications included the removal of a premature stop codon present in the transregulator and the domestication of KRAB to remove an unwanted recognition site for BsaI. The final syntax contained 2 constructs (BBa_K4213049); the TetR-KRAB expressing platform (pNOS:TetR:NLS:KRAB:tNOS) next to the Reporter gene expressing platform (tetO7:pTriple:Venus:tNOS).
A consistent problem
The two constructs were built using the Golden Braid assembly method. For the experiment, we compared inhibition abilities of the normal TetR, next to the same NLS (pNOS:TetR:NLS:tNOS) and reporter system, to the improved device. The constructs were first assembled and we planned to introduce them to N. benthamiana leaves through Agroinfiltration to achieve transient expression, where the leaf samples would be collected after 4 days of agroinfiltration to be submerged for 1 minute in Tetracycline dissolved in Sodium Citrate. Finally, the samples would be observed under a confocal microscope 0, +4 and +8 hours post incubation, whereupon we expected to observe increased fluorescence on account of the conformational change of the transregulator in the presence of Tetracycline.
Unfortunately, due to the lack of time, we were not able to conduct the experiment and confirm our literature review. So, we leave our documentation here, for future iGEM Teams to see and complete by providing the necessary results that we could not.
References
[1] Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjørn, S. P., Givskov, M., & Molin, S. (1998b, June). New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and Environmental Microbiology, 64(6), 2240–2246. https://doi.org/10.1128/aem.64.6.2240-2246.1998
[2] Lupo, A., Cesaro, E., Montano, G., Zurlo, D., Izzo, P., & Costanzo, P. (2013d, June 1). KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions. Current Genomics, 14(4), 268–278. https://doi.org/10.2174/13892029113149990002
[3] Yin, J., Yang, L., Mou, L., Dong, K., Jiang, J., Xue, S., Xu, Y., Wang, X., Lu, Y., & Ye, H. (2019c, October 23). A green tea–triggered genetic control system for treating diabetes in mice and monkeys. Science Translational Medicine, 11(515). https://doi.org/10.1126/scitranslmed.aav8826
[4] Gatz, C., & Quail, P. H. (1988e, 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
[5] Lu, J., Wu, T., Zhang, B., Liu, S., Song, W., Qiao, J., & Ruan, H. (2021e, 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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 761
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 761
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 761
- 1000COMPATIBLE WITH RFC[1000]
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