Part:BBa_K3128032
COMP : Clickable OmpX
Clickable OmpX is an outer membrane protein whose C and N terminal in the intracellular domain. It can be used as a scaffold if an unnatural amino acid is to be introduced. This can be done through the presence of the amber stop codon TAG in the third loop OmpX. With a specific tRNA, an amino acid functionalized with an azide will be integrated, which can be used for the SPAAC click chemistry reaction with DBCO / DIBO functional groups. Once expressed, this biobrick will serve as a positive control to check the click for biobrick BBa_K3128015 et BBa_3128016 during the expression test
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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
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