Difference between revisions of "Part:BBa K3128033"
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==Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne confirmation:==
 
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Click-iT ™ Alexa Fluor ™ 488 sDIBO alkyne was used to confirm whether Ompx is in memebrane and if the unnatural amino acid is incorporated into OmpX. This fluorophore is used to check the reaction of the click. If the unnatural amino acid is present, the fluorophore should "click" on the OmpX transmembrane protein and remain there. This can be analyzed with fluorescence microscopy
 
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Here are the results obtained on unprocessed BL21 (1) and the results obtained for BL21 cotransformed with the 2 vectors (2).
 
  
  

Revision as of 20:20, 11 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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