Part:BBa_K3128020:Experience

Team Grenoble-Alpes 2019

mBACTH

Materials and Methods

Bacterial Strain

The assays are made with streptomycin resistant BTH101 E.Coli strain, which are cya- bacteria.
In this strain, the endogenous adenylate cyclase gene has been deleted in order to obtain a bacterium that is unable to produce endogenous cAMP, thus avoiding the presence of potential false positives and making the system more sensitive.

Design of the plasmids

For the mBACTH, as three biobricks have to be inserted in the bacterium to constitute the entire system, genetic constructions have been made in order to co-transform only two compatible plasmids:

pOT18-Nlc contains OmpX gene fused to the T18 sub-part and the NanoLuciferase gene under the control of the plac promoter; it has an ampicillin resistant gene and the pMB1 replication origin.
pOT18-Nlc contains NanoLuciferase reporter for BACTH assay and OmpX WT protein fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter.
pOT25 contains OmpX gene fused to the T25 subpart. It has a kanamycin resistant gene and the p15A replication origin.
pOT25 contains OmpX WT protein fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter.
Those constructs will constitute the negative condition that will reveal the background noise of the initial mBACTH system.

pOT18-Nlc-ZIP is similar to pOT18-Nlc with the addition of a leucine-zipper sequence between the OmpX signal peptide and the OmpX gene.
pOT18-Nlc-ZIP contains NanoLuciferase reporter for BACTH assay and OmpX WT protein fused with LZ and T18 subpart of Bordetella Pertussis AC under constitutive promoter.
pOT25-ZIP is similar to pOT25 with the addition of a leucine-zipper sequence between the OmpX signal peptide and the OmpX gene.
pOT25-ZIP contains OmpX WT protein fused with LZ and T25 subpart of Bordetella Pertussis AC under constitutive promoter.
Those constructs will constitute the positive condition that will reveal how the signal increases when both sub-parts are brought together with the mBACTH.

Transformation

For the assay with the membrane BACTH, BTH101 are co-transformed either with
pOT18-Nlc and pOT25 plasmids : AC sub-parts fused to OmpX  : negative condition,
or pOT18-Nlc-ZIP and pOT25-ZIP plasmids : Leucine Zipper mediated reconstitution of AC : positive condition.

The assay

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.

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 20µLworkto 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 for the values of time above 90 min 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 free sub-parts : negative condition and the Leucine Zipper: positive condition from around 210 minutes.

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.

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 induction time, 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 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_K1492002.
The expression of BBa_K1492002 was induced by adding arabinose then the unnatural amino acid is added in the medium.

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 with the endogenous proteins of E. coli without the necessary molecular system.
It will also show if the Dalexia 488 conjugated DIBO group can interact with a bacteria that have not the unnatural amino acid in it's proteins.
For that we have incubated Bl21 in the presence of pAzF : the unatural amino acid.
The lack of fluorescence shows that there is no click chemistry reaction.
It has been verified that the amino acid does not integrate and interact with Dalexia 488 conjugated DIBO group 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 make pAzF interact with other proteins that could have a TAG codon (cytosolic or membrane) which would lead to different non-specific click reactions.
If Dalexia 488 conjugated DIBO group diffuse into the cytosol their will be a high fluorescent signal. If their is a signal that ressemble the BL21 E.coli + pAzF control it means that Dalexia 488 conjugated DIBO group don't diffuse into the bacterium. It has been verified that even with the presence of pEVOL-pAzF, the non-natural amino acid does not bind to other proteins, so it means that the Dalexia 488 conjugated DIBO group doesn't diffuse into the bacterium. (Figure 2)



Those controls shows that :
-Dalexia 488 conjugated DIBO don't interact with protein that have not the pAzF
-Dalexia 488 conjugated DIBO don't diffuse in the bacterium
It means that, if we have a superior fluorescent intensty in the following assay then the Dalexia 488 conjugated DIBO has interact with a protein that have integrate the unatural amino acid and is located in the external membrane of the bacterium.

Test for click and membrane expression of COMP

Positive Control

BL21 E.coli + pEVOLE-pAzF + pAzF + COMP:
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 protein COMP can incorporate the pAzF and thus realize the reaction of the click.
The fluorescent intensity indicates that in the presence of pEVOL-pAzF and of the non-natural amino acid, the COMP protein which is in the external membrane, react with the Dalexia 488 conjugated DIBO (reaction click) which proves the pAzF integrates into the COMP structure.
It also show the difference of flurescent intensity between the negative and positive control.
(Figure 3)

Click on COMP fused with sub-part of AC

BL21 E.coli + pEVOLE-pAzF + pAzF + COMB-T18:
COMP fused to the sub-part T18 of adenylate cyclase, was expressed in the presence of pAzFs and aminoacyl-tRNA synthetase (via co transforming a plasmid containing the sequence of COMP-T18 and pEVOL-pAzF) in order to show that the COMP-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.(Figure 4)

BL21 E.coli + pEVOLE-pAzF + pAzF + COMP-T25:
COMP fused to the sub-part T25 of adenylate cyclase, was expressed in the presence of pAzFs and aminoacyl-tRNA synthetase (via co transforming a plasmid containing the sequence of COMP-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:
COMP linked to the T25 subunit of adenylate cyclase is capable of being expressed, directed towards the membrane and realize the reaction of click chemistry. (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.
Those 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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