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Team:TU Eindhoven/Project/Characterization/Click Coli
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<div class="col_700_2 float_r"> | <div class="col_700_2 float_r"> | ||
| + | <h2>Click Coli</h2> | ||
<figure style="float:right;margin-right:0;"> | <figure style="float:right;margin-right:0;"> | ||
<img id='Fig1' src="https://static.igem.org/mediawiki/2014/0/06/TU_Eindhoven_Chemical_Structure_of_TAMRA-DBCO.png" width="500" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | <img id='Fig1' src="https://static.igem.org/mediawiki/2014/0/06/TU_Eindhoven_Chemical_Structure_of_TAMRA-DBCO.png" width="500" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> | ||
| - | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Chemical structure of DBCO-PEG <sub>4</sub> -5/6-TAMRA.</figcaption> | + | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Chemical structure of DBCO-PEG<sub>4</sub>-5/6-TAMRA.</figcaption> |
</figure> | </figure> | ||
| - | + | ||
| - | <p>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- | + | <p>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-PEG<sub>4</sub>-5/6-TAMRA (Figure 1), have been reacted with the incorporated p-azido-L-phenylalanine (pAzF).</p> |
| + | |||
| + | <p>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-PEG<sub>4</sub>-5/6-TAMRA, the relative fluorescence of each cell has been measured using FACS. | ||
| + | <br><br> | ||
| + | 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.</p> | ||
<h4>Bibliography</h4> | <h4>Bibliography</h4> | ||
Revision as of 19:06, 13 October 2014
Click Coli
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.
