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Team:TU Eindhoven/Manual
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<p>In order to use the clickable outer membrane proteins developed by iGEM team TU Eindhoven 2014 you will need both the plasmid containing the mutated membrane protein (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1492000">BBa_K1492000</a> or <a target="_blank" href="http://parts.igem.org/Part:BBa_K1492001">BBa_K1492001</a>) in a pET29a vector and the plasmid containing the orthogonal tRNA system (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1492002">BBa_K1492002</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1223014">BBa_K1223014</a>), in a pEVOL plasmid we ordered ours <a target="_blank" href="http://www.addgene.org/31186/">here</a>. For <i>E. coli</i> and other gram-negative bacteria we recommend using our COMPx protein as this has been thoroughly tested in our lab and can be mutated at the C- or N-termini to display different oligo-peptide tags, should you want to. For gram-positive bacteria another membrane protein should be used, as COMPx is specific for the outer membrane of E. coli, which gram-positive bacteria don’t have. Also check that your host does have an amber suppression system, as this is essential for the orthogonal tRNA system.</p> | <p>In order to use the clickable outer membrane proteins developed by iGEM team TU Eindhoven 2014 you will need both the plasmid containing the mutated membrane protein (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1492000">BBa_K1492000</a> or <a target="_blank" href="http://parts.igem.org/Part:BBa_K1492001">BBa_K1492001</a>) in a pET29a vector and the plasmid containing the orthogonal tRNA system (<a target="_blank" href="http://parts.igem.org/Part:BBa_K1492002">BBa_K1492002</a> and <a target="_blank" href="http://parts.igem.org/Part:BBa_K1223014">BBa_K1223014</a>), in a pEVOL plasmid we ordered ours <a target="_blank" href="http://www.addgene.org/31186/">here</a>. For <i>E. coli</i> and other gram-negative bacteria we recommend using our COMPx protein as this has been thoroughly tested in our lab and can be mutated at the C- or N-termini to display different oligo-peptide tags, should you want to. For gram-positive bacteria another membrane protein should be used, as COMPx is specific for the outer membrane of E. coli, which gram-positive bacteria don’t have. Also check that your host does have an amber suppression system, as this is essential for the orthogonal tRNA system.</p> | ||
| + | <p>Any chemical compound containing the Dibenzocyclo-octyne (DBCO) moiety can be clicked to the COMP, however it is wise to include a hydrophilic spacer, such as PEG4, between the DBCCO and the functional group to increase solubility of the compound and create some distance from the bacterial membrane and the possibly reactive group.</p> | ||
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Revision as of 21:57, 17 October 2014
How to use our tool
Introduction
In order to use the clickable outer membrane proteins developed by iGEM team TU Eindhoven 2014 you will need both the plasmid containing the mutated membrane protein (BBa_K1492000 or BBa_K1492001) in a pET29a vector and the plasmid containing the orthogonal tRNA system (BBa_K1492002 and BBa_K1223014), in a pEVOL plasmid we ordered ours here. For E. coli and other gram-negative bacteria we recommend using our COMPx protein as this has been thoroughly tested in our lab and can be mutated at the C- or N-termini to display different oligo-peptide tags, should you want to. For gram-positive bacteria another membrane protein should be used, as COMPx is specific for the outer membrane of E. coli, which gram-positive bacteria don’t have. Also check that your host does have an amber suppression system, as this is essential for the orthogonal tRNA system.
Any chemical compound containing the Dibenzocyclo-octyne (DBCO) moiety can be clicked to the COMP, however it is wise to include a hydrophilic spacer, such as PEG4, between the DBCCO and the functional group to increase solubility of the compound and create some distance from the bacterial membrane and the possibly reactive group.
