Team:TU Eindhoven/Project/Characterization/Click Coli

Click Coli

Verification of Protein Expression

Now that the plasmid design has been verified using the sequencing results it is time to check whether the plasmids result in the wanted protein expression and more importantly if the SPAAC reaction works on a bacterial cell membrane. 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 FACS for sorting with DBCO-TAMRA).

When a comparison between the different DBCO to COMP ratios is made in which only the amount of cells is varied thus the amount of COMP available in the sample. A significant difference in intensity can be seen (Figure 3). However the peak of the fluorescence does not shift, all the peaks are around 2*10³. Therefore, it is concluded that all COMPx is labeled with DBCO-PEG₄-TAMRA. In future similar experiments, the DBCO to COMP ratio should be 31.5.

As can be read in 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 been 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 labelling).

For reasons that have been described previously, similar future experiments will be performed using COMPx, with a DBCO concentration of 30μM, a DBCO/COMP ratio of at least 31.5 and an incubation time of 1 hour.

Reacting COMPx with larger DBCO functionalized Molecules

It has been verified that the cells express COMPx successfully and that small DBCO-molecules can be reacted on these membrane proteins. To verify the applicability of this system as a general clickable system, it is necessary to investigate whether larger DBCO functionalized molecules can also be reacted on COMPx. It was thought that after reaction with 10kDa polyethyleneglycol (PEG)-tails the cells would be bigger and that this increase in size would be detectable using the forward scatter data obtain from FACS. Although the results after an incubation time of 1 hour suggest a slight increase in size, from the results after an incubation time of 6 hours can be concluded that no significant change in size can be detected using forward scatter data. (Figure 5)

To verify the reaction of larger DBCO functionalized molecules and to provide more insight into the optimal concentration for this reaction, bacterial cells expressing COMPx have been incubated with different concentrations of DBCO-PEG(5kDa)-TAMRA (100pM-100μM). (Figure 6) It can be seen that despite the relatively high concentrations (100μM), the characteristic plateau does not show. To further investigate this, bacterial cells expressing COMPx have been incubated with either 3.16μM or 31.6μM pure TAMRA. Since pure TAMRA does not react with the cells, no fluorescence should be detected after washing the cells. After performing FACS, this can be indeed seen when the cells are incubated with 3.16μM pure TAMRA. When the cells are incubated with 31.6μM pure TAMRA, fluorescence can be detected. This indicates that the wash-steps are not sufficient to fully wash away the excessive/unreacted DBCO molecules, explaining the absence of the plateau. Despite the insufficient wash-steps, it can be concluded that larger DBCO functionalized molecules indeed show significant reaction with COMPx (see protocol FACS for sorting with DBCO-PEG (10kDa)).

Bibliography

Click Chemistry Tools, B. T. (n.d.). Products: TAMRA DBCO. Retrieved July 23, 2014, from
https://www.clickchemistrytools.com/products/click_chemistry_toolbox/azide_cycloalkyne_click_chemistry/cyclooctynes/fluorescent_probes/tamra-dbco-prob/

JenaBioscience. (n.d.). DBCO Reagents - PEGylation Reagents. Retrieved July 6, 2014, from
http://www.jenabioscience.com/cms/en/1/catalog/1840_pegylation_reagents_.html