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Revision as of 20:05, 16 September 2015
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 NeonGreen & NanoLuc BRET Pair
To analyze the performance of the NeonGreen & NanoLuc containing COMBs, we cloned both membrane proteins separately in the pET-Duet vector as well as in a single vector. Both for the vector containing either NanoLuc or NeonGreen and the vector containing both, we analyzed whether the click reaction occured and whether bioluminescence and fluorescence could be measured. An overview of our achievements is shown below.
OmpX-NeonGreen
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Verification of the DBCO 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 non-natural amino acid (pAzf). The click reaction for OmpX-mNeonGreen was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much higher fluorescence intensity than the negative controls (purple and blue).
This indicates that the click-reaction occurs.
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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 high peak at a wavelength of 517 nm. Wehereas 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
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Verification of the DBCO 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 non-natural amino acid (pAzf). The click reaction for OmpX-NanoLuc was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much 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 (NanoLuc). Bioluminiscence is measured using a spectrophotometer, the spectrum is shown in the figure below. After addition of furamizine OmpX-NanoLuc shows a high peak characteristic for NanoLuc, indicating NanoLuc's presence within the cells. bioluminiscence picture
OmpX-NeonGreen & OmpX-NanoLuc
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Verification of the DBCO click reaction
& localization in the outer membrane
The click reaction should be specific and therefore should only take place on the non-natural amino acid (pAzf). The click reaction for OmpX-mNeonGreen & OmpX-NanoLuc was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much higher fluorescence intensity than the negative controls (purple and blue).
This indicates that the click-reaction occurs.
-
Verification of fluorescence
OmpX-NeonGreen's presence in the cells was verified by measuring NeonGreen's presence in the platereader. The peak with its maximum at 517 nm indicates that NeonGreen is indeed present. Cells without NeonGreen showed no response in fluorescence measurement, indicating that the peak originates from OmpX-NeonGreen.
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Verification of bioluminescence
& BRET
OmpX-NanoLuc's & OmpX-NeonGreen's presence in the cells were tested by adding furimazine to the cells and measuring bioluminescence with the spectrophotometer. The figure below shows that OmpX-NanoLuc shows a high 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.
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 & fluorescence was visible.
OmpX-LgBiT
-
Verification of the DBCO 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 non-natural amino acid (pAzf). The click reaction for OmpX-LgBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much higher fluorescence intensity than the negative controls (purple and blue).
This indicates that the click-reaction occurs.
OmpX-SmBiT
-
Verification of the DBCO 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 non-natural amino acid (pAzf). The click reaction for OmpX-SmBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much higher fluorescence intensity than the negative controls (purple and blue).
This indicates that the click-reaction occurs.
NanoBiT
-
Verification of the DBCO click reaction
& localization in the outer membrane
The click reaction should be specific and therefore should only take place on the non-natural amino acid (pAzf). The click reaction for OmpX-LgBit & OmpX-SmBit was tested using DBCO-PEG4-TAMRA, a fluorescent dye.
As can be seen in the figure below, the bacteria which had pAzF available (green) showed much 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.