To make sure that the OmpX-T18 and OmpX-T25 are expressed in the external membrane, OmpX fusion proteins have been muted to be able to integrate an unnatural amino acid in one of their extracellular loops by implementing the amber stop codon TAG. A specific tRNA can then add an azido-modified amino acid to the protein, these modified proteins are called COMPs.
The azido group of the protein reacts with a DIBO group, the reaction allows to click the extracellular DIBO to the functionnalized biosensor (COMP) protein.
COMPs are fused with T18 or T25 subparts and have to be expressed at the external membrane of the bacteria.
To ensure this, microscopy observations have been done with an Dalexia 488 conjugated DIBO group.
Fluorescent microscopy observations of the COMP, COMP-T18 and COMP-T25 clickable proteins show surface labelled bacteria indicating that a the recombinante proteins are expressed at the external membrane of E. coli.
See the the experiments below

The bioluminescence intensity produced by the NanoLuciferase enzyme is determined.
Several experimental conditions are tested using decreasing amount of bacterial culture (100µL, 25µL, 5µL and 1µL) at OD600nm = 0.6 : respectively 48E+06 CFU, 12E+06 CFU, 24E+05 CFU and 48E+04 CFU .
In addition, times of induction are tested from 0 to 360 minutes with 30 minutes increments.
Cultures of the different recombinant bacteria are incubated overnight at 18°C under shaking in order to induce an optimal COMPs proteins production [http://2015.igem.org/Team:TU_Eindhoven cf Team Eindhoven 2015].
The low temperature allows a native protein folding and membrane insertion to avoids as much as possible the formation of inclusion bodies.

Then cultures are diluted at OD600nm = 0,4 and let to grow to OD 600nm = 0.6 before induction.
The induction is performed by addition of 0,5 mM IPTG and 2mM of ATP for different periods of time. Bacteria are incubated at 37°C under shaking (180 rpm) to allow an optimal NanoLuciferase production.

After induction, 1, 5, 25 or 100µL of bacteria are distributed in a 96 wells black NUNC plate (ThermoFisher) and the Nano-Glo® Luciferase Assay assay from Promega® is performed (More informations) :
“Prepare the desired amount of reconstituted Nano-Glo® Luciferase Assay Reagent by combining one volume of Nano-Glo® Luciferase Assay Substrate with 50 volumes of Nano-Glo® Luciferase Assay Buffer.For example, if the experiment requires 10 mL of reagent, add 200μl of substrate to 10 mL of buffer.”
Then the amount of bioluminescence is measured using a luminometer by recording Relative Luminescence Units (RLU).

Several measures are made in the same well in order to reduce incertitude induced by the luminometer.
In order to test the reproducibility of our measures the means of 3 differents experiments with 3 measurements per well are calculated.
Data are expressed as the mean +/- standard deviation.

Several controls are performed:
∅ IPTG, ∅ ATP : To check the promoter leakage without any induction.
∅ IPTG, 2 mM ATP :To check if the addition of extracellular ATP helps the production of cAMP and to check if addition of ATP modifies the promoter leakage.
0.5 mM IPTG, ∅ ATP : To check if adding extracellular ATP is needed for protein expression.
0.5 mM IPTG, 2 mM ATP : Is the experimental condition, it correspond to the measure at 360min.

Results

The mBACTH following results are obtained with 5µL of bacteria at OD600nm = 0.6 : 24E+05 CFU.
With 1µL (48E+04 CFU), the bioluminescence intensity was too low and the measurement were not discriminant enough.
Above 25µL of bacteria (12E+06 CFU), the signal was quickly saturated when the induction time increased and the luminometer could not record workable measures.
5µL (24E+05 CFU) is a good compromise, it’s enough to have a discriminant signal and sensitive enough to work as a small drop in our NeuroDrop device.
iGEM Grenoble-Alpes device NeuroDrop is designed for the use of small volumes of biological sample like drops. Proving that 5µL of bacteria are enough to detect a significant difference in bioluminescence intensity between negative and positive conditions result was a challenge that we have overcome. Other reagents (see the full system) will be added to the drop of bacteria and its volume should not exceed 20µL to allow its automatic moving on the surface of the device.


Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the mBACTH generated with either
BBa_K3128018 and BBa_K3128017 : AC sub-parts fused to OmpX  : negative condition,
or BBa_K3128026 and BBa_K3128027 : Leucine Zipper mediated reconstitution of AC : positive condition.
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted to each measurements.

Using positive control strain, we measured 1.48E+06 RLU of bioluminescence produced in the 0.5 mM IPTG condition compared to 9.02E+05 in the condition without IPTG and without ATP, indicating that IPTG increase slightly the transcription.
Additionally, with 2.55E+0,6 RLU of bioluminescence produced in the condition without IPTG and 2mM ATP condition compared to 9.02E+05 in the without IPTG and without ATP condition, it seems that ATP have a significant* effect on transcription.
This was expected because of the lack of ATP in the periplasm of the bacteria. Thereby, adding a great amount of ATP in the medium able to diffuse in the periplasm help the cAMP production by the periplasmic adenylate cyclase.
Obviously, those observations do not prove anything but give clues on the way the system operates.
* A T test was done between both control and led to a p-value below 0.01.


Luminescence production over time of induction for the negative condition strain (red curve) and the positive condition strain of the mBACTH assay (blue curve).
Area of the significant* difference between both curves is highlighted in yellow.
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted to each measurements.
* A T test was done for the values of time above 210 min and led to a p-value below 0.05.

From 0 to 120 minutes of induction time, the bioluminescence produced by the two strains is similar. At 120 minutes, the two curves start to split and give rise to a significant difference between the two strains from around 210 minutes the negative condition strain compared to the Leucine Zipper_positive condition .

The discrepancy keeps increasing upon time of induction, thus highlighting the efficiency of the amplification signal thanks to the signalling cascade and the strong reporter gene.

Conclusion

There is a significant difference between the negative and the positive condition of the mBACTH assay,
suggesting that a Bacterial Adenylate Cyclase Two-Hybrid can be successfully performed in the periplasm of bacteria which property is required for the sensing and detection of extracellular molecules.

Click on COMP

The following parts parts were use to characterise the click on COMP aspect of this part
BBa_K3128033 correspond to the positive control : COMP
BBa_K3128019 correspond to the test with COMP fused with T18
BBa_K3128020 correspond to the test with COMP fused with T25
More informations on click-chemistery here

The experiment

The purpose of these experiments was to confirm that COMP, COMP-T18 and COMP-T25 are expressed in the external membrane and to validate that the unnatural amino acid is incorporated into OmpX. The clickable fluorophore (Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne) is used to rapidly check the click reaction on COMP. We verified the expression o on BL21 co-transformed with a vector that contains COMP, COMP-T18 or COMP-T25 and a second vector pEVOL-pAzF: BBa_K1492002.
The expression of BBa_K1492002 was induced by adding arabinose (0.2%) then the unnatural amino acid p-Azidp-L-phenylalanine (pAzF) (1mM) is added in the medium for 15 hours at 18°C.
Bacteria was incubated with 25mM or 30mM of Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne for 10 minutes.

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 present 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.

Negative Controls

BL21 E.coli + pAzF:
In this experiment, we wanted to assert that the unnatural amino acid can not integrate in endogenous proteins of E.coli without the necessary molecular system namely a amber codon insertion in the target protein.
It will also show the background due to non-specific fixation of the Alexia Fluor 488 conjugated DIBO at the surface of the bacteria.
For that purpose we have incubated Bl21 in the presence of pAzF : the unnatural amino acid.
The very low level of fluorescence indicates that there are no non-specific click reaction. These data suggest that the unnatural amino acid is not spontaneously incorporated into proteins that do not contain the appropriate mutation and that the DIBO moiety does not clicked on proteins expressed at the cell surface.


Figure 1 : Fluorescent-conjugated DIBO labelling of BL21 E coli in presence of pAzF.

BL21 Ecoli + pEVOL-pAzF + pAzF In this experiment, we wanted to check if the pAzF could be incorporated thanks to the amynoacyl tRNA-transferase activity, to random cytoplasmic or membrane proteins that have a TAG codon which would lead to unwanted click reactions.
If Alexia Fluor 488 conjugated DIBO group would diffuse into the cytosol a high fluorescent signal would be recorded. If the recorded signal would be similar to the one obtained above (figure 1), namely the BL21 E.coli + pAzF control it would suggest that the DIBO group doesn't diffuse into the bacterium.
As shown in the picture (figure 2) no signal is obtained in the experimental condition suggesting that even in presence of pEVOL-pAzF, the non-natural amino acid is not incorporated into unmodified bacterial proteins.

Figure 2 : Fluorescent-conjugated DIBO labelling of BL21 E coli containing pEVOL-pAzF cultured in presence of pAzF.

The data presented figure 1 and 2 are control conditions showing that :
-Alexia-Fluor 488 conjugated DIBO doesn't click on proteins that have not incorporated the pAzF
-Alexia-Fluor 488 conjugated DIBO doesn't diffuse and accumulate into the cytoplasm.

Test for click and membrane expression of COMP

Positive Controls

BL21 E.coli + pEVOLE-pAzF + pAzF + COMP:
Mutated OmpX (COMP) was expressed in the presence of both pAzF and aminoacyl-tRNA synthetase (via transformation of a plasmid containing the sequence of COMP and pEVOL-pAzF). in order to show that the pAzF can be incorporated into the protein COMP on which the DIBO group could be clicked.
Figure 3 shows that most of the bacteria are fluorescent after incubation with Alexia-Fluor 488 conjugated DIBO. These data indicate that bacteria expressed the COMP protein (pAzF containing OmpX) in their external membrane and that the DIBO group can be clicked on the azido group of the pAzF.

Figure 3 :Fluorescent-conjugated DIBO labelling of BL21 E coli transformed with BBa_K3128032 and pEVOL-pAzF cultured in presence of pAzF.

BL21 E.coli + pEVOLE-pAzF + pAzF + COMB-T18 or COMB-T25:
COMPs either fused to the T18 sub-part of adenylate cyclase or to the T25 sub-part, were expressed in the presence of pAzFs and aminoacyl-tRNA synthetase (via co transforming a plasmid containing pEVOL-pAzF and either the sequence of COMP-T18 or the one of COMP-T25).

Figure 4 shows fluorescent bacteria in both conditions revealing that:
The pAzF is incorporated in the COMP-T18 and COMP-T25 proteins ,
These recombinant proteins are properly expressed into the external membrane of the bacteria
The fluorescent conjugated DIBO group can be clicked on the pAzF incorporated in COMP.

Figure 4 : Fluorescent-conjugated DIBO labelling of BL21 E coli either BBa_K3128019 or BBa_K3128020 and pEVOL-pAzF cultured in presence of pAzF.

Conclusion

Figure 5 : Comparative of all flurescence assays tested above

These data establish the proof of concept of our biological system.
Culturing recombinant bacteria transformed with PEVOL-pAzF and a plasmid contain the COMP sequence fused to sub-parts of the adenylate cyclase in presence of pAzF allows the expression of COMP-T18 and COMP-T25 at the accurate localisation in the membrane of the bacteria.
We also succeeded to click a fluorescent-conjugated DIBO alkyne group on these recombinant proteins indicating that DIBO conjugated specific aptamers could be successfully clicked to the COMPs. That will create our biosensor system.
This biosensor is versatile and very powerful since any DIBO-conjugated ligand (aptamers, nanobodies, proteins…) can be fixed on COMPs to recognize its specific target.

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