Part:BBa_K3128033:Experience

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Applications of BBa_K3128033

Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne confirmation:
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 Here are the results obtained on unprocessed BL21 (Figure 1) and the results obtained for BL21 cotransformed with the 2 vectors (Figure 2).

BL21 E.coli + pAzF:
In this experiment we wanted to assert that the unnatural amino acid can not integrate with the endogenous proteins of E. coli without the necessary molecular system. and for that we have incubated Bl21 in the presence of pAzF The absence of fluorosis shows that there is no membrane click chemistry reaction. It has been verified that the amino acid does not integrate without the presence of the total expression system (Figure 1)

BL21 Ecoli + pEVOL-pAzF + pAzF :

In this experiment we want to check if in the presence of amynoacyl tRNA-transferase will allow pAzF to integrate into other membrane proteins which would lead to different non-specific click reactions. It has been verified that even with the presence of pEVOL-pAzF, the non-natural amino acid does not bind to other membrane proteins, so we can be sure that there will be no false positives regarding the reaction of the click. (Figure2).

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