Part:BBa_K4213045

pNOS:TetR:KRAB:tNOS

Engineering Cycle 2: Third Iteration: Upgrading Repressing abilities

Design

It was pointed out to us by Prof. Aikaterini Kalliampakou that tetracycline-controlled systems can be quite leaky. After careful literature review, we learnt that ‘leaky’ gene expression is a common and rather persistent problem in synthetic biology and often contributes to the poor performance of a genetic circuit ^[1]. This led us to the investigation and utilization of the Tet toolbox ^[2].

Improving our repressor with a transregulator

In more detail, 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) ^[3], enhancing the inhibition abilities of the complex by adding another mechanism of silencing ^[4]. Since our design already incorporated TetR, we just needed to add KRAB next to the repressor as this is the supposed working syntax ^[5]. For this we had to remove the already present stop codon by inducing a mutation. Furthermore, after the conducted optimization of the part, we had to domesticate it by inducing a silent mutation, in order to remove an unwanted recognition site for BsaI.

The final syntax contained mVenus as, again, we wanted to spot protein expression with the help of fluorescent microscopy. This led to a final construct containing pNOS:Venus:TetR:NLS:KRAB:tNOS.

Build

After almost three weeks of cloning experiments, we managed to confirm the assembly of the abovementioned construct by diagnostic digestion and visualization through gel electrophoresis.

Figure 1: Diagnostic Digestion of: 1 and 3 “pNOS-Venus-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 2056 and 513) 2 and 5“pNOS-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 1342 and 513) Positive results: 2, 4 and 5; 1 MW.

Test

We repeated agroinfiltration in Nicotiana benthamiana leaves and our samples were prepared with the previously mentioned counterstain for better visualization of the nucleus for observation under the confocal microscope.

Learn

With the images presented below, we confirm the expression of our transregulator as well as its localization in the nucleus.

Figure 2: Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.

References

[1] Ho, J. M. L., Miller, C. A., Parks, S. E., Mattia, J. R., & Bennett, M. (2020c, December 8). A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria. Nucleic Acids Research, 49(5), e25–e25. https://doi.org/10.1093/nar/gkaa1179

[2] Berens, C., & Hillen, W. (2004c). Gene Regulation By Tetracyclines. Genetic Engineering: Principles and Methods, 255–277. https://doi.org/10.1007/978-0-306-48573-2_13

[3] Lupo, A., Cesaro, E., Montano, G., Zurlo, D., Izzo, P., & Costanzo, P. (2013e, 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

[4] Yin, J., Yang, L., Mou, L., Dong, K., Jiang, J., Xue, S., Xu, Y., Wang, X., Lu, Y., & Ye, H. (2019d, 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

[5] Sigl, R., Ploner, C., Shivalingaiah, G., Kofler, R., & Geley, S. (2014b, May 19). Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR). PLoS ONE, 9(5), e97764. https://doi.org/10.1371/journal.pone.0097764

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 192
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]