‘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.

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 protein1 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.

Figure 1. Part C0040 of Registry.

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 silencing3. 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 likely4, the addition of the Simian Virus 40 NLS5 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).

Figure 2. The TetR-KRAB Expressing platform next to the Reporter Gene Expressing platform.

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 (BBa_K4213028), for future iGEM Teams to see and complete by providing the necessary results that we could not.