Part:BBa_K4213041

TetO7:pTriple:Venus:tNOS(stuffer)

Engineering Cycle 3: First Iteration: Deciding on Regulatory Elements

Design

The Second Reporter System

Beginning with the promoter, we opted for the Cauliflower Mosaic Virus 35S promoter (p35s) as this is arguably the most well-studied and experimentally used regulatory component with activity in plant cells ^[1]. With abundant information available on its individual functional domains, we could incorporate Tet Operator (TetO) sequences within the promoter, placing the second construct under the influence of TetR. Therefore, we introduced three repeats of TetO to p35s and set them in close proximity to the TATA box, based on the design of pTriple Op promoter ^[2]. Given that we planned to test both TetR and TetR-KRAB for their repressive abilities, we also introduced seven direct repeats of the Tet Response Element (TRE) upstream of the distal part of p35s, based on the design of pTight (also known as pTetO7)(3). With the distance between the TIS and the modified TRE being under 3.000 bases, this was the best fit for TetR-KRAB’s long range repressive power ^[3]. Continuing on, both the protein and the terminator of choice would be the same as the first reporter system so we ended up with a final construct containing mod-p35s:Venus:tNOS.

Build

After two 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 “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387.

Test

We repeated agroinfiltration in Nicotiana benthamiana leaves but this time we were not able to visualize our constructs in our University’s confocal microscope, due to malfunctions. Fortunately, we were able to measure the fluorescence of our prepared samples with the help of the Plate Reader.

Learn

With the results presented below we confirm the expression mVenus and, therefore, the functionality of our modified p35s. Next step was the expression of the pht1 gene.

Figure 2: Fluorescence of different leaf samples measured under the Plate Reader.

References

[1] Amack, S. C., & Antunes, M. S. (2020c, December). CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. Current Plant Biology, 24, 100179. https://doi.org/10.1016/j.cpb.2020.100179

[2] Gatz, C., Frohberg, C., & Wendenburg, R. (1992e, May). Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. The Plant Journal, 2(3), 397–404. https://doi.org/10.1046/j.1365-313x.1992.t01-37-00999.x

[3] Deuschle, U., Meyer, W. K., & Thiesen, H. J. (1995c, April). Tetracycline-reversible silencing of eukaryotic promoters. Molecular and Cellular Biology, 15(4), 1907–1914. https://doi.org/10.1128/mcb.15.4.1907

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 353
  Illegal PstI site found at 347
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 353
  Illegal NheI site found at 1515
  Illegal PstI site found at 347
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 353
  Illegal XhoI site found at 34
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 353
  Illegal PstI site found at 347
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 353
  Illegal PstI site found at 347
  Illegal NgoMIV site found at 28
  
• 1000
  COMPATIBLE WITH RFC[1000]