Team:TU Eindhoven/Manual

Click Coli Manual

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 do not 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 DBCO and the functional group to increase solubility of the compound and create some distance from the bacterial membrane and the possibly reactive group.

Step by step instructions:

1. Make sure that the COMPx gene is present in a pET29a(+) vector under control of the lac-repressor. This can be done by the use of the restriction enzymes EcoRI/XbaI and NotI. Protocols can be found here for digestion and ligation.
2. Make sure the tRNA system is in a pEVOL vector under control of an arabinose promoter.
   NOTE: Check that both vectors have different origins of replication and antibiotic resistances when using other vectors for expressing COMPx and the tRNA system
3. Prepare LB-agar plates containing kanamycin and chloramphenicol (or your preferred antibiotics when using other vectors). Our protocol can be found here.
4. Perform a double transformation to introduce both vectors into the host strain.
5. Pick a couple of colonies and use these for protein expression.
6. You should now have sample of cells expressing COMPx which can be used to click with DBCO containing molecules (here is a general protocol for DBCO-PEG-TAMRA).
   
   You can order your DBCO molecules ready made from numerous suppliers online OR you could synthesize them yourself if they contain a reactive group such as a primary amine