Team:TU Eindhoven/Overview

Overview Click Coli

Below a quick overview is given of the mechanism behind Click Coli. This system enables the formation of a covalent bond of any DBCO functionalized molecule onto protein anchors that are expressed on the outer membrane of E. coli. If you wish to use this click system, please take a look at our step-by-step manual.

1. Two plasmids are transformed into E. coli. One contains a orthogonal tRNA system, which is the engineered Methanocaldococcus jannaschii tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair orthogonal to E. coli. [1] The other one contains the sequence that encodes for a mutated outer membrane protein, hereafter named Clickable Outer Membrane Protein (COMP).










2. A closer look at the plasmid encoding for the Clickable Outer Membrane Protein (COMP) – either COMPx or COMPy - reveals two important regions. By site directed mutagenesis an amber stop codon (TAG) is introduced into the sequence for the outer membrane protein, which will be used by the orthogonal system. Also a Human influenza hemagglutinin tag (HA tag) is introduced at the C-terminus of the outer membrane protein for evaluation of expression purposes.













3. Before protein expression, the unnatural aminoacid p-AzidoPhenylalanine (pAzF) is added to E. coli. This unnatural amino acid is able to permeate into the cytoplasm, when it is added to the growth medium of the E. coli bacteria.







4. During protein expression, the orthogonal tRNA system only enables the incorporation of the unnatural amino acid pAzF at the place given by the Amber Stop codon (TAG), but is unable to lead to the incorporation of any of the 20 common amino acids at the TAG codon.











5. After the proteins are synthesized, they will be transported to the bacterium’s outer membrane. There they will be folded in the correct form, ensuring that the unnatural amino acid pAzF and the HA-tag are located on the outside of the cell.











6. A Clickable Outer Membrane Protein has an HA-tag on the outside of the bacterium’s membrane. This HA-tag can be used to evaluate the level of expression of the proteins by adding anti-HA antibodies with a fluorescent marker, see Verification Protein Expression. However, the click reaction of the pAzF still needs to be evaluated.











7. A molecule that is able to be clicked onto the outer membrane requires to be Dibenzocyclooctyl (DBCO) functionalized. A fluorescent dye, linked by a spacer made of Polyethylene glycol (PEG) to a DBCO group, is added to the E. coli bacterium enriched with Clickable Outer Membrane Proteins.








8. The DBCO group and the pAzF are able to form a covalent bond in the so called Strain Promoted [3+2] Azide-Alkyne Cycloaddition (SPAAC). No further reagents are required for this highly efficient and selective SPAAC reaction.










9. The SPAAC reaction ensures the covalent bonding of a Clickable Outer Membrane Protein to a fluorescent marker. The level of fluorescence can be determined by FACS in order to assure the click reaction has taken place. In the end the bacterium is covered by the desired molecules, which are covalently bound to its outer membrane by means of the Clickable Outer Membrane Proteins.

Bibliography

[1] Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S., Wang, L., & Schultz, P. G. (2002). Addition of-Azido--phenylalanine to the Genetic Code of. Journal of the American Chemical Society, 124(31), 9026-9027.