Team:TU Eindhoven/Results





Results



To analyze our Clickable Outer Membrane Proteins, we have carried out many experiments. Our experimental approach towards these COMBs has been described in our Experimental Approach. For a more in-depth review of our results, take a look at our raw data page. Here, we hope to shortly present our achievements with our COMB Prototypes.


COMB Prototype I: The mNeonGreen & NanoLuc BRET Pair



To analyze the performance of the mNeonGreen & NanoLuc containing COMBs, we cloned both membrane proteins separately in the pETDuet-1 vector as well as both in a single vector. For both the vector containing either NanoLuc or mNeonGreen and the vector containing both proteins, we analyzed whether the click reaction occured and whether bioluminescence and fluorescence could be measured. An overview of our achievements is shown below.

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.






COMB Prototype II: NanoBiT



For NanoBiT, we undertook the same approach as for our BRET pair: we cloned the parts together into a single pETDuet-1 vector as well as separately into different vectors. For all of these constructs, we determined whether the click reaction occured, and if bioluminescence and fluorescence was visible.

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.




Verification of the DNA-click



The first step taken after obtaining working COMBs was to verify whether the SPAAC Click reaction occured with DBCO-functionalized DNA. To verify this, DNA was clicked to the outer membrane proteins in the same ratio as with the TAMRA test. After having clicked complementary DNA to the COMBs, the presence of complementary DNA could be verified through the addition of complementary, fluorescent DNA strands. To verify whether both complementary strands clicked to the COMBs, the COMBs with clicked DNA strands were incubated simultaneously with two different fluorescent complementary strands. The first oligo was Cy5-labeled, the second was TAMRA-labeled (see the Figure below). Fluorescence of the individual cells was subsequently measured using the FACS.
  • DBCO-DNA Click for the first COMB prototype &
    The figure below is a three dimensional visualization of the dimerization of DNA-fluorophores to the DNA clicked on the cell. The fluorescence intensity of one DNA-fluorophore, Cy5, is deplicted in the figure below on the y-axis. The fluorescence intensity of the other DNA-fluorophore, TAMRA, is depicted in the figure below on the x-axis. The brightness of each dot is a measure for the relative occurence of labelled bacteria.
    From the figure shown below, it can be deduced that there is a strong correlation between Cy5 and TAMRA intensity if the bacteria are clicked (see A). The negative control where bacteria are not clicked show less correlation and have a lower density (see B). These results thus indicate that the fluorescently labeled DNA is not washed away for bacteria which have been incubated with DBCO-modified DNA, indicating that the DBCO-modified indeed clicks on the bacteria.
  • DBCO-DNA Click for the second COMB prototype &
    The figure below is a three dimensional visualization of the dimerization of DNA-fluorophores to the DNA clicked on the cell. The fluorescence intensity of one DNA-fluorophore, Cy5, is deplicted in the figure below on the y-axis. The fluorescence intensity of the other DNA-fluorophore, TAMRA, is depicted in the figure below on the x-axis. The brightness of each dot is a measure for the relative occurence of labelled bacteria.
    From the figure shown below, it can be deduced that there is a strong correlation between Cy5 and TAMRA intensity if the bacteria are clicked (see A). The negative control where bacteria are not clicked show less correlation and have a lower density (see B). These results thus indicate that the fluorescently labeled DNA is not washed away for bacteria which have been incubated with DBCO-modified DNA, indicating that the DBCO-modified indeed clicks on the bacteria.




Complementary DNA





To verify whether proximity indeed yield a intracellular response, we devised testing our COMBs with complementary DNA. Through the use of DNA strand displacement. A critical parameter for our device is the amount of complementary DNA used, as too little DNA will yield no response. Moreover, too much DNA will also yield no response, since the probability that a single DNA strand binds two COMBs decreases as the amount of DNA increases (see the figure below).
If too much DNA is added to the COMBs, the probability that a single DNA strand binds two COMBs is very low. This means that the presence of DNA does not induce proximity between two COMBs, such that presence of DNA does not result in a measurable signal.

To find out how much DNA should be added to the COMBs to obtain a measurable signal, we conducted numerous complementary DNA assays. The results we obtained, however, were too inconsistent for us to base any conclusions on. Some of the results we obtained with our complementary DNA assays are shown below:
  • Complementary DNA assay with NanoBiT
    The NanoBiT construct was tested for a wide range of concentrations. The assay was carried out in duplo with a negative control. Even though the deviation between data points seemed quite high, early in the assay a significantly higher intensity was measured in the low nanomolar range. We failed, however, to replicate this result.
    The first NanoBiT complementary assay. The figure shows a significantly higher level of bioluminescence at approximately 4nM.
    The second NanoBiT complementary assay. The figure shows a significantly higher level of bioluminescence at approximately 2nM.
    The final NanoBiT complementary assay. This complementary assay served as a negative control and was conducted with cells which were not clicked.
  • Complementary DNA assay with mNeonGreen and NanoLuc
    The mNeonGreen + NanoLuc prototype was tested for a wide range of concentrations. The assay was carried out in duplo with a negative control. No trend could be seen in our data, possibly due to the relatively low amount of cells we used: when we excitated mNeonGreen using a laser, no mNeonGreen could be measured, leading to the indication that the expression of mNeonGreen was too low.
    The first mNeonGreen & NanoLuc complementary assay. No trend can be seen.
    The second mNeonGreen & NanoLuc complementary assay. No trend can be seen in the figure, possibly due to the low amount of cells used for measurement.
    The final mNeonGreen & NanoLuc complementary assay. This complementary assay served as a negative control and was conducted with cells which were not clicked. No trend could be seen.
  • Incubation of bacteria with clicked DNA and complementary DNA in the spectrofluorometer
    We also measured the cells with complementary DNA in the spectrofluorometer with a wide range of negative controls. These negative controls included cells without clicked DNA and negative controls without complementary DNA. Measurement was performed for five cycles and the average is plotted in the figure below for the assay with mNeongreen and NanoLuc. For the measurement with NanoBit three cycles were measured and averaged. No significant ratiometric change could be detected for the mNeonGreen and NanoLuc prototype and no significant increase in intensity could be detected for the NanoBiT prototype.
    Complementary DNA was measured in the spectrofluorometer with a wide range of negative controls. No ratiometric change could be detected.
    The second mNeonGreen & NanoLuc complementary assay. No trend can be seen in the figure, possibly due to the low amount of cells used for measurement (5.3*10^6 cells/mL).




Site-Directed Mutagenesis



We did not succeed in replicating the results from our complementary DNA assay. A hypothesis we had for failing to find a solid ratiometric change for the BRET system and no change in the bioluminescence signal was the fact that we used too few cells. In addition to the fact that we used a low amount of cells, the first insert in the pETDuet-1 vector suffers from a lower level of expression than the second insert. To obtain equimolar expression, we mutated the Ribosome Binding Site of both prototypes to become weaker. The SDM itself seemed to be succesful, as we obtained colonies. However, subsequent tests with the mutated vectors did show less than satisfactory results: for our NanoBiT prototype, we failed to measure any bioluminescence at all and for our mNeonGreen+NanoLuc prototype, we could not find a ratiometric change in our complementary DNA assay.

Future Testing



In order to test whether the system will relay significantly better signals when brought into close proximity, several actions can be taken to fine-tune the assay with complementary DNA. Expression rates of the outer membrane proteins, concentration of complementary DNA strands and concentrations of cells/mL during the measurements can be optimized.