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COMB Prototype I: The mNeonGreen & NanoLuc BRET Pair

OmpX-mNeonGreen

• Verification of the click reaction and whether the protein is located in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-mNeonGreen was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.


• Verification of fluorescence
  The fluorescent protein mNeongreen should be intact, this is tested by laser excitation. The emission spectrum, as can be seen below, shows a peak at a wavelength of 517 nm. Whereas the negative control, OmpX-SmBit & OmpX-Lgbit, do not show any peaks at all. Indicating that mNeongreen is indeed present and intact in the cell.

OmpX-NanoLuc

• Verification of the click reaction and whether the protein is located in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-NanoLuc was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.


• Verification of bioluminescence
  The presence of the luciferase, OmpX-NanoLuc, in the cells is tested by adding its substrate (Nano-Glo). Bioluminiscence is measured using a spectrophotometer, the spectrum is shown in the figure below. After addition of the Nano-Glo substrate (which contains furimazine) OmpX-NanoLuc shows a peak characteristic for NanoLuc, indicating NanoLuc's presence within the cells.

OmpX-mNeonGreen & OmpX-NanoLuc

• Verification of the click reaction & localization in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-mNeonGreen & OmpX-NanoLuc was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.
• Verification of fluorescence
  OmpX-mNeonGreen's presence in the cells was verified by measuring mNeonGreen's presence in the platereader. The peak with its maximum at 517 nm indicates that mNeonGreen is indeed present. Cells without mNeonGreen showed no response in fluorescence measurement, indicating that the peak originates from OmpX-mNeonGreen.
• Verification of bioluminescence & BRET
  OmpX-NanoLuc's & OmpX-mNeonGreen's presence in the cells were tested by adding Nano-Glo substrate to the cells and measuring bioluminescence with the spectrophotometer. The figure below shows that OmpX-NanoLuc shows a peak characteristic for NanoLuc, indicating NanoLuc's presence within the cells. Moreover, the spectrogram shows a distinct shoulder near 517 nm, the emission wavelength of mNeongreen. Since no laser was used, excitation of mNeongreen can only be accomplished by NanoLuc so BRET occured. This signal is measured when no click reaction is performed on the complex, meaning that this can be seen as the background noise of the sensor.




• Dimerization of DNA-fluorophores to clicked DNA
  After verifying the click of DBCO-Tamra to all of our constructs the next step can be made. This time DBCO-DNA is clicked, subsequently two types of DNA-fluorophores are added to anneal. One fluorophore, Cy5, is deplicted in the figure below on the axis called "Alexa 633A" (which is its filter) showing its intensity. The other fluorophore, Tamra, is deplicted in the figure below on the axis called "PE-A" (which is its filter) showing its intensity. As one can see in figure A there is a strong correlation between bacteria (dots) both having a high intensity Cy5 and Tamra fluorescence. Figure B represents the negative control where the DBCO-DNA is not added, so no clicked occured.

COMB Prototype II: NanoBiT

OmpX-LgBiT

• Verification of the click reaction and whether the protein is located in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-LgBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.

OmpX-SmBiT

• Verification of the click reaction and whether the protein is located in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-SmBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.

NanoBiT

• Verification of the click reaction & localization in the outer membrane
  The click reaction should be specific and therefore should only take place on the unnatural amino acid (pAzf). The click reaction for OmpX-LgBit & OmpX-SmBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye functionalized with DBCO.
  As can be seen in the figure below, the bacteria which had pAzF available (green) showed higher fluorescence intensity than the negative controls (purple and blue).
  This indicates that the click reaction occurs.
• Verification of bioluminescence
  When both OmpX-SmBiT & OmpX-LgBiT are present in the cell they can come together and form a complex. In both domains form a complex (NanoBit) bioluminiscence can be measured. For future sensor use this can be seen as background noise. The combination of OmpX-SmBiT and OmpX-LgBiT showed a bright peak, whereas cells expressing either OmpX-SmBiT or OmpX-LgBiT showed no such peak, indicating that the split luciferase is present in the cells and works.