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Team:TU Eindhoven/Project/Characterization/Click Coli
From 2014.igem.org
Click Coli
Verification of Protein Expression
Now that the plasmid design has been verified using the sequencing results and the SPAAC reaction has been verified using UV-VIS and LCMS, it is time to check whether the plasmids result in the wanted protein expression. To verify the expression of Clickable Outer Membrane Protein X (COMPx) and Y (COMPy), fluorescent labelled dibenzocyclooctyne (DBCO) groups, DBCO-PEG4-5/6-TAMRA (Figure 1), have been reacted with the incorporated p-azido-L-phenylalanine (pAzF).
To determine the optimal conditions for the DBCO-azido reaction, the recombinant expressed COMPs have been reacted with two different concentrations of DBCO-TAMRA, 5 μM and 30 μM. These concentrations have been adapted from commercially available fluorescent labelled DBCO kits. (Click Chemistry Tools) Furthermore, the bacterial cells have been incubated for two different time spans, one hour and six hours, which have been adapted from commercially available DBCO products (Click Chemistry Tools; JenaBioscience). To further optimize the conditions, the ratios of DBCO to COMP have been varied, ratios of 31.5, 63 and 126.1 have been used. After incubation with DBCO-PEG4-5/6-TAMRA, the relative fluorescence of each cell has been measured using FACS.
When the influence of the concentration is being analyzed, it can be seen that for both COMPx and COMPy the fluorescence is significantly higher when the cells are incubated with 30μM of DBCO compared to 5μM of DBCO. (Figure 2) Therefore, in similar future experiments a concentration 30μM DBCO will be used.
The influence of the incubation time with DBCO shows different results for COMPx and COMPy. Analyzing the results for COMPx, it can be seen that longer incubation time causes a slight shift of the entire curve to higher fluorescence for both 5μM and 30μM. (Figure 2: A versus C) For COMPy, it can be seen that besides the slight shift of the entire curve to a higher fluorescence, also the shape of the curve changes. After an incubation time of 6 hours, there are less cells with a lower fluorescence and more cells with a higher fluorescence. (Figure 2: B versus D) Keeping in mind that it is beneficial that more DBCO groups have reacted with a COMP, but shorter incubation times are more practical, it has been decided that for similar future experiments, the incubation time should be at least 1 hour (see protocol DBCO-PEG4-TAMRA).
COMPx reacted with different DBCO-PEG4-TAMRA to COMP ratios.
When a comparison between the different DBCO to COMP ratios is made, no significant difference can be seen. (Figure 3) Therefore, it has been concluded that in future similar experiments, the DBCO to COMP ratio should be at least 31.5.
As can be read under Background Membrane Anchors, COMPy has been adapted from an already available BioBrick and COMPx has been adapted from a membrane protein that has not previously described within the iGEM competition. Therefore a direct comparison between both membrane displaying systems is interesting to see. Conclusions can already be drawn when comparing the FACS results with DBCO-TAMRA of COMPx and COMPy (Figure 2: A versus B and C versus D). But the results have been further expanded by treating both membrane proteins with Mouse anti Hemagglutinin (HA) antibodies (400 nM), since both systems express an HA-tag. From these results can be concluded that COMPx has a better expression rate than COMPy and/or the azide of COMPx is better accessible. Therefore, similar future experiments will be performed using cells expressing COMPx (see protocol Antibody labeling).
