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| - | <h2>Results: SPAAC on Cells</h2> | + | <h2>Membrane Anchors</h2> |
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| - | <span>Verification of protein expression</span> | + | <span>Gene Design</span> |
| - | <p class="para">Now that our 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).</p> | + | <p class="para">In short, both membrane proteins had a TAG codon introduced for the Non Natural Amino Acid and a HA-tag for detection methods. For INPNC the TAG codon and the HA-tag were introduced at the C terminus of the protein (figure 1). The CPX proteins already contained a peptide library part at the N-terminus so the TAG codon was introduced in that part. This has been done by using Site Directed Mutagenesis. The HA-tag was introduced at the C-terminus (figure 2). After modification the proteins were renamed to Clickable Outer Membrane Protein x (COMPx), originally CPX, and COMPy, originally INPNC.</p> |
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| - | <img src="https://static.igem.org/mediawiki/2014/0/06/TU_Eindhoven_Chemical_Structure_of_TAMRA-DBCO.png"> | + | <img src="https://static.igem.org/mediawiki/2014/4/4b/TU_Eindhoven_COMPy_design.png"> |
| - | <figcaption>Figure 1. Chemical structure of DBCO-PEG4-5/6-TAMRA.</figcaption> | + | <figcaption>Figure 1. Gene design COMPy (INPNC)</figcaption> |
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| - | <p class="para">To determine the optimal conditions for the DBCO-azido reaction, the recombinant expressed COMPs have been reacted with two different concentrations of DBCO-TAMRA, 5μM 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.
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| - | 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.
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| - | 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.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/3/34/TU_Eindhoven_COMPx%2BCOMPy_1h%2B6h.jpg"> | + | <img src="https://static.igem.org/mediawiki/2014/4/43/TU_Eindhoven_COMPx_design.png"> |
| - | <figcaption>Figure 2. FACS results of bacterial cells that have expressed a COMP reacted with either no, 5μM or 30 μM DBCO-PEG4-TAMRA. A. COMPx incubated with DBCO for 1 hour. B. COMPy incubated with DBCO for 1 hour. C. COMPx incubated with DBCO for 6 hours. D. COMPy incubated with DBCO for 6 hours.</figcaption> | + | <figcaption>Figure 2. Gene design COMPx (CPX)</figcaption> |
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| - | <p class="para">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.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/e/ea/TU_Eindhoven_Overlay_different_ratios.jpg">
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| - | <figcaption>Figure 3. FACS results of bacterial cells that have expressed COMPx reacted with different DBCO-PEG4-TAMRA to COMP ratios.</figcaption>
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| - | <p class="para">As can be read under ???, 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, we thought it was very interesting to make a direct comparison between both membrane displaying systems. 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.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/d/df/TU_Eindhoven_Overlay_COMPx_%2B_COMPy.jpg">
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| - | <figcaption>Figure 4. FACS results of bacterial cells expressing A. COMPx or B. COMPy treated with Mouse Anti HA antibodies (400 nM).</figcaption>
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| - | <p class="para">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 (at least) 1 hour.
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| - | <span>Reacting COMPx with bigger DBCO molecules</span>
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| - | 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 bigger DBCO 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)</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/9/9f/TU_Eindhoven_Overlay_DBCO_PEG_10_kDa_1h_%2B_6h.jpg">
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| - | <figcaption>Figure 5. FACS results of the forward scatter of bacterial cells expressing COMPx, which have been incubated with DBCO-PEG(10kDa) for A. 1 hour or B. 6 hours.</figcaption>
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| - | <p class="para">To verify the reaction of bigger DBCO 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 bigger DBCO-molecules indeed show significant reaction with COMPx.</p>
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| - | <img src="https://static.igem.org/mediawiki/2014/9/9e/TU_Eindhoven_Final_peg5.jpg">
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| - | <figcaption>Figure 6. FACS results of the bacterial cells expressing COMPx that have been incubated with DBCO-PEG(5kDa)-TAMRA (below), a schematic representation is provided above.</figcaption>
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| - | <span>Bibliography</span>
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| - | [1] 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/
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| - | [2] Macey, M. G. (2007). Cell Sorting by Flow Cytometry. In Flow Cytometry: Principles and Applications (pp. 257 - 276). Humana Press.
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