Revision as of 10:32, 13 October 2019

Team Grenoble-Alpes 2019

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IMPROVE OF THE BACTERIAL ADENYLATE CYCLASE TWO HYBRID

Purpose

The goal here is to prove that an Outer-membrane bacterial two hybrid (mBATCH) can be generated and can be functional by improving two biobricks from the original BACTH system:
BBa_K1638004 : T18 domain of adenylate cyclase from Bordetella pertussis
BBa_K1638002 : T25 domain of adenylate cyclase from Bordetella pertussis

In order to do this, 4 biobricks are created:
K3128017 : OmpX Wild-Type (WT) protein fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter
K3128018 : OmpX WT protein fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter
These two biobricks constitute the negative condition of the mBACTH (free sub-parts condition). OmpX proteins are fused to the adenylate cyclase sub-parts at their N-terminal ends. The fusion protein move freely in the bacterial outer membrane, but they are not forced to get closer by any mean.
The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.
The signal measured here is considered as background noise.

K3128026 : OmpX WT protein fused with Leucine-zipper (LZ) and T18 sub-part of Bordetella Pertussis AC under constitutive promoter
K3128027 : OmpX WT protein fused with Leucine-zipper and T25 sub-part of Bordetella Pertussis AC under constitutive promoter
These two biobricks constitute the positive condition of the mBACTH (Leucine Zipper condition). OmpX proteins are fused to the AC subparts at their N-terminal ends, and a leucine-zipper sequence is added between the [https://parts.igem.org/wi

Click on COMP

The experiment

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 COMP,COMP-T18 or COMP-T25 and a second vector pEVOL-pAzF : BBa_K149202. The expression of BBa_K1492002 was induced by adding arabinose and in the presence of the unnatural amino acid

Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne confirmation:
Click-iT ™ Alexa Fluor ™ 488 sDIBO alkyne was used to confirm whether COMP,COMP-T18 and COMP-T25 are 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 COMP transmembrane protein and remain there.
This was 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).

pAzF
The unnatural amino acid used to incorporate an azide in the anchor proteins is p-Azido-L-phenylalanine (pAzF). pAzF is a photocrosslinker which can be incorporated in any protein, irrespective of its size or sequence, by a tRNA synthetase/tRNA pair and the amber codon TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslinks interacting proteins.

Controls

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).

Test for click and membrane expression of COMP

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).

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)

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)

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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