Composite
COMP

Part:BBa_K3128033:Experience

Designed by: Fahd TIBOURTINE   Group: iGEM19_Grenoble-Alpes   (2019-09-29)
Revision as of 21:50, 11 October 2019 by Fahd (Talk | contribs)


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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)

Click Test Figure 1.png


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).

Click Test Figure 2.png

BL21 E.coli + pEVOLE-pAzF + pAzF + COMP:
Mutated COMP was expressed in the presence of pAzF and aminoacyl-tRNA synthetase (via transformation of a plasmid containing the sequence of OmpX and pEVOL-pAzF) in order to show that the protein OmpX can incorporate the pAzF and thus realize the reaction of the click. The presence of flurence indicates that it was possible to verify that in the presence of pEVOL-pAzF and of the non-natural amino acid, the COMP protein expresses itself in the same membrane and arrived at a chemical reaction click which proves the pAzF integrates into the ompX structure without any problem (figure 3).

Click Test Figure 3.png

BL21 E.coli + pEVOLE-pAzF + pAzF + COMB-T18:
The fusion protein,mutated OmpX related to the sub unit T18 of adenylate cyclase, was expressed in the presence of pAzFs and aminoacyl-tRNA synthetase (via co transforming a plasmid containing the sequence of OmpX-T18 and pEVOL-pAzF) in order to show that the OmpX-T18 protein can incorporate the pAzF and thus realize the reaction of the click It has been shown that in the presence of pEVOL-pAzF and the unnatural amino acid, the fusion protein: COMP linked to the T18 subunit of adenylate cyclase is capable of being expressed, directed towards the membrane and realize the reaction of click chemistry without problem (figure 4)

Click Test Figure 4.png

BL21 E.coli + pEVOLE-pAzF + pAzF + COMP-T25:
The fusion protein,COMP related to the sub unit T25 of adenylate cyclase, was expressed in the presence of pAzFs and aminoacyl-tRNA synthetase (via co transforming a plasmid containing the sequence of OmpX-T25 and pEVOL-pAzF) in order to show that the COMP-T25 protein can incorporate the pAzF and thus realize the reaction of the click It has been shown that in the presence of pEVOL-pAzF and the unnatural amino acid, the fusion protein: OmpX mutated linked to the T25 subunit of adenylate cyclase is capable of being expressed, directed towards the membrane and realize the reaction of click chemistry without problem (figure 5)

Click Test Figure 5.png



conclusion
Figures 1 and 2 show that "Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne" does not entre inside bacteria, confirming that the markings observed for COMP, COMP-T18 and COMP-T25 are external markings . these results confirm also the need to have a complete expression system (PEVOL-pAzF + plasmid contain COMP sequence) to ensure the reaction of chemistry click

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