Part:BBa_K4497017

MESA GFPnb-L1-tTA One of eight receptor parts that can be used the the MESA receptor system. The receptors are made of a nanobody receptor for either GFP or mCherry, followed by two different link lengths, and an intracellular domain of either the transcription factor tTA or the protease TEV. tTA is cut off the receptor on contact with the TEV protease. The release of the transcription factor can be used to induce reporters or other expression in the cell of interest.

MESA System Overview

Figure 5. Overview over Possible Combinations of MESA System. (A): Our designs incorporate two different nanobodies against mCherry (M) and GFP (G). Two different linker lengths to the transmembrane domain were created 1 (SEFSGGN) and 2 (SEFGGDYKDDDDNGGSGGSGGSGGSGGGTG). Finally, two different internal functions can be used: Protease (TEV), Transcription Factor tTA (TA). This leads to 8 different combinations in total: M1TA, M1TEV, M2TA, M2TEV, G1TA, G1TEV, G2TA, G2TEV. (B) Two examples for possible combination - left: homodimeric combination M1TA & M1TEV - right: heterodimeric combination M1TA & G1TEV.

These are all the others MESA parts:

• G1TA: BBa_K4497017
• G1TEV: BBa_K4497018
• M1TA: BBa_K4497019
• M1TEV: BBa_K4497020
• G2TA: BBa_K4497021
• G2TEV: BBa_K4497022
• M2TA: BBa_K4497023
• M2TEV: BBa_K4497024

Cytometer Measurements

We tested all the possible combinations of two of our MESA system, inducing them with the corresponding synthetic ligand dimer (2xmCherry, 2xGFP or GFP-mCherry). The activation of each receptor pair was measured using our tTA inducable miRFP680 reporter (BBa_K4497030)

Figure 9. Reporter activation of the MESA system upon ligand binding in HEK293T cells. Different MESA component combinations were tested without (blue) and with (red) ligand addition, M1TA M1TEV (A), M2TA M2TEV (B), G1TA G1TEV (C), G2TA G2TEV (D), M1TA G1TEV (E), G1TA M1TEV (F), M2TA G2TEV (G), G2TA M2TEV (H). For each combination three transfection ratios of TF to TEV were tested: 6x, 12x and 24x. The APC signal was corrected by dividing APC MFI (Median) by the BFP MFI (Median) of each sample. Ligand was added 24 h after transfection, cytometer measurements were performed 48 h after transfection. Bar plots show the median of one sample with a total of at least 3000 APC positive gated events counted.

Unfortunately, none of the screened MESA combinations show a significant increase in reporter signaling on ligand addition. Schwarz et al. [4] showed reporter induction of 2x and more for their best MESA combinations. It might be that our chosen incubation times, both before adding ligand and before measuring are not optimal. It might be that the cells need more or less time with or without ligand to give a better signal differentiation. Additionally, we might not have screened the optimal plasmid ratios or created suboptimal linker lengths. A more broad screen with more constructs might reveal an optimal condition that we missed. It has to be added that the shown cytometry analysis results only resemble one transfection sample, so one biological replicate. While this is not enough for obtaining significant results, we were hoping to find a promising MESA component and ligand combination in this scanning experiment.

Figure 7. GFP and mCherry fluorescence signals of mCherry (A) and GFP MESA (B) components in HEK293T cells. The Mean Fluorescence Intensity (MFI) of mCherry and GFP was obtained of at least 30000 cells per sample. MFI was calculated using the geometric mean. Control- & Control+: MFI of cells containing no MESA parts, without and with added ligand. MFI of cells containing one MESA part: mCherry MESA (A) or GFP (B), Transcriptionfactor (TF), TEV polymerase (TEV) with linkers 1 or 2 and added ligand.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 454
  Illegal XbaI site found at 559
  Illegal PstI site found at 349
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 454
  Illegal PstI site found at 349
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 454
  Illegal BamHI site found at 103
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 454
  Illegal XbaI site found at 559
  Illegal PstI site found at 349
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 454
  Illegal XbaI site found at 559
  Illegal PstI site found at 349
  
• 1000
  COMPATIBLE WITH RFC[1000]