Team:TU Eindhoven/Click Reaction

From 2014.igem.org

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<span>Verification of protein expression</span>   
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        <span>Click Coli: expanding the chemical toolbox for bacteria</span>   
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      <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>
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      <p class="para">A fundamental problem in utilizing genetically modified bacteria is their limited ability to survive under non-natural conditions, such as the harsh conditions in industrial reactors and the immune system in the human body. We report a “Plug-and-Play” system allowing the introduction of chemical anchors on cell membranes, subsequently used to attach a functional coating. The anchors consist of azidophenylalanine, which couples covalently with molecules containing the strained alkyne DBCO in a so-called "click" reaction. We obtained proof of concept that our developed Clickable Outer Membrane Proteins (COMP) enable this fast and effective click reaction. We apply it to create a safe “clicked-on” coating allowing E.coli to be used in the human body for healthcare purposes. Furthermore, microfluidic devices have been designed, ensuring increased control over the click reaction and single cell coating. We believe our “Plug-and-Play” system is a versatile tool providing numerous possibilities for engineering bacteria on outer membranes.
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Revision as of 05:52, 13 September 2014

Project Description

Results: SPAAC on Cells

Click Coli: expanding the chemical toolbox for bacteria

A fundamental problem in utilizing genetically modified bacteria is their limited ability to survive under non-natural conditions, such as the harsh conditions in industrial reactors and the immune system in the human body. We report a “Plug-and-Play” system allowing the introduction of chemical anchors on cell membranes, subsequently used to attach a functional coating. The anchors consist of azidophenylalanine, which couples covalently with molecules containing the strained alkyne DBCO in a so-called "click" reaction. We obtained proof of concept that our developed Clickable Outer Membrane Proteins (COMP) enable this fast and effective click reaction. We apply it to create a safe “clicked-on” coating allowing E.coli to be used in the human body for healthcare purposes. Furthermore, microfluidic devices have been designed, ensuring increased control over the click reaction and single cell coating. We believe our “Plug-and-Play” system is a versatile tool providing numerous possibilities for engineering bacteria on outer membranes.