Difference between revisions of "Part:BBa K3128033"

Latest revision as of 22:02, 12 October 2019

COMP under constitutive promoter

Clickable OmpX is an outer membrane protein whose C and N terminals are in the intracellular domain. It can be used as a scaffold, a non-natural amino acid must be introduced. This can be done through the presence of the stop codon TAG amber in the third loops OmpX.. With a specific tRNA, an azide functionalized amino acid can be integrated, which can be used for the SPAAC click chemistry reaction with DBCO / DIBO functional groups Sequence and Features

Usage and Biology

The presence of the amber stop codon TAG will enable it to integrate the non-natural amine at the third loop of its scaffolding role for future functional variants of the cyclooctin group. It has been shown during the different experiments that we have done to introduce the amino acid should not be the secondary structure of OmpX. You can find all the information related to the introduction of non-natural amino acid in the structure of our site (https://2019.igem.org/Team:Grenoble-Alpes)

Characterization

Once the expression protocol has been performed in the presence of the amino acid functionalized with the azide group, it has been shown that ompX binds with covalent manicure to almost everything as long as it contains a group functionalized with DIBO. The bond is ensured by the reaction of the click chemistry.

We performed expression tests in the presence of clickable fluorophore (Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne) on BL21 co-transformed with a vector that contains mutated ompX and a second vector pEVOL-pAzF (BBa_K1492002). The expression was induced by adding arabinose and in the presence of the unnatural amino acid