Team:TU Eindhoven/Overview

Overview Click Coli

Below a quick overview is given of the mechanism behind Click Coli. This system enables the covalent bonding 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 plasimids are transformed into E. coli. One contains a orthogonal tRNA synthetase, which is the engineered Methanocaldococcus jannaschii tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair orthogonal to Escherichia 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) has been 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 the cytoplasm, when it is added to the growth medium of the E. coli bacteria.







4. During protein expression, the orthogonal synthase 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 bacteria’s outer membrane. There they will be folded in the correct manner, 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 cell’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, it is the pAzF on the outside that is essential for the click reaction.











7. The molecule that is desired to be clicked onto the outer membrane requires to be Dibenzocyclooctyl (DBCO) functionalized. In figure 7, a fluorescent dye, TAMRA, linked by a spacer made of Polyethylene glycol (PEG) to a DBCO group is added to the E. coli bacteria 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 click ensures the covalent bonding of every 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 bacteria cell is covered by the desired molecules, which are covalently bound to its outer membrane by 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.