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.