Part:BBa_K1638002:Experience

Team Grenoble-Alpes 2019

Complete article here

IMPROVE OF THE BACTERIAL ADENYLATE CYCLASE TWO HYBRID

Purpose

The goal here is to prove that a Outermembrane 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 signal peptide of OmpX -to express the recombiant protein in the external membrane- and OmpX gene, in order to force the physical closeness of OmpX proteins.
Leucine zippers are peptides which contain a hydrophobic leucine residue at every seventh position. They are able to dimerize through interactions between their helices.
This is a strategy to force physical closeness. Hence the AC activity will be restored through the interaction of both subparts and will induce the cAMP dependant signalling cascade.

The reporter gene used in the system is the NanoLuciferase enzyme present in the BBa_K3128001 under a cAMP inducible CAP-dependent lactose promoter.
Two major factors affect this promoter :
IPTG, known to have a positive effect on the transcription of the gene by removing the lac repressor from the DNA.
CAP, known to have a positive effect on the transcription when it binds cAMP by helping the fixation of RNA-polymerase on the DNA
To be able to bind the CAP sites on the promoter, the CAP protein has first to interact with a cAMP molecule. As soon as two cAMP-CAP complexes are bound to the CAP sites, the RNA Polymerase initiates the transcription.



ATP is not naturally present in large amount in the periplasm of the bacteria, thereby it has to be added in the bacteria medium to enhance its periplasm diffusion and to be available for the adenylate cyclase catalytic reaction.

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

To compare the efficiency of the BACTH system created with the initial BACTH biobricks BBa_K1638004 (containing the T18 subpart) and BBa_K1638002 (containing the T25 subpart), quantification results in BTH101 strain are needed.
pJT18 contains the T18 sub-part ; it has an ampicillin resistant gene and the pMB1 replication origin.
pJT18 contains T18 subpart of Bordetella Pertussis AC under constitutive promoter.
pJT25-Nlc contains the T25 sub-part and the NanoLuciferase gene under the control of the plac promoter. It has a kanamycin resistant gene and the p15A replication origin.
pOT25-Nlc contains NanoLuciferase reporter for BACTH assay and T25 subpart of Bordetella Pertussis AC under constitutive promoter.
Those constructs will be the negative condition that show the background noise of the initial mBACTH system.

pJT18-ZIP is similar to pJT18-Nlc with the addition of a Leucine Zipper sequence fused at the end of T18.
pJT18-ZIP contains T18 subpart of Bordetella Pertussis AC fused with Leucine-zipper under constitutive promoter.
pJT25-Nlc-ZIP is similar to pJT25-Nlc with the addition of a Leucine Zipper sequence fused at the end of T25.
pJT25-Nlc-ZIP contains NanoLuciferase reporter for BACTH assay and T25 subpart of Bordetella Pertussis AC fused with Leucine-zipper under constitutive promoter.
Those constructs will be the positive condition that show how the signal increases if both sub-parts are brought together with the BACTH.

Genetic constructions of pJT18, pJT25-Nlc, pJT18-ZIP and pJT25-Nlc-ZIP plasmids used to test the cytoplasmic BACTH in BTH101 strain.

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 be the negative condition that show 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 be the positive condition that show how the signal increases if both sub-parts are brought together with the mBACTH.

Genetic constructions of pOT18-Nlc, pOT25, pOT18-Nlc-ZIP and pOT25-ZIP plasmids used to test the membrane BACTH in BTH101 strain.

Transformation

For the assay with the BACTH, BTH101 are co-transformed either with pJT18 and pOT25-NlC plasmids : negative condition,
or pJT18-ZIP and pJT25-Nlc-ZIP plasmids : positive condition.
For the assay with the mBACTH, BTH101 are co-transformed either with pOT18-Nlc and pOT25 plasmids : negative condition,
or pOT18-Nlc-ZIP and pOT25-ZIP plasmids : positive condition.

Classic cytoplasmic BACTH and mBACTH assay

To test the two different BACTH systems, the bioluminescence intensity produced by the NanoLuciferase enzyme is analyzed.
Several conditions are tested with 100µL, 25µL, 5µL and 1µL of bacteria 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) 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.”
The bioluminescence is then observed with a luminometer by measuring Relative Luminescence Units (RLU).

Several measures are made in the same well in order to reduce the incertitudes 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 measured with the calculation of standard deviation for each.

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 normal condition, it correspond to the measure at 360min.

Results

Cytoplasmic BACTH assay

The cytoplasmic BACTH, the following results are obtained with 5µL of bacteria (24E+05 CFU).

Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the BACTH generated with either
pJT18 and pJT25-Nlc : free sub-parts : negative condition,
or pJT18-ZIP and pJT25-Nlc-ZIP : Leucine Zipper : positive condition.
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.

With 7,48E+06 RLU of bioluminesce produced in the 0,5 mM IPTG condition compared to 6,02E+06 in the condition without IPTG and 2mM ATP, it seems that IPTG increase slightly the transcription.
Additionally, with the same produced bioluminescence<u> between the without IPTG and 2mM ATP and without IPTG and without ATP conditions, <u>ATP appears to have no effect on transcription.
These two observations were expected because of the large amount of ATP already present in the cytoplasm of the bacteria saturating the adenylate cyclase.
Obviously, those observations don’t prove anything but give clues on the way the system operates.


Luminescence production over time of induction for the negative condition strain (yellow curve) and the positive condition strain of the BACTH assay (purple 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 from the measurements.
* A T test was done for the values of time above 90 min and led to a p-value below 0.05.

This figure indicates that the production of luminescence by the two conditions are similar for the first 60 minutes of induction. Then, a significant difference between the two strains is observed from 90 minutes to 360 minutes of induction (yellow zone) with a continuous increase gap between both condition as time go on. This discrepancy highlights the efficiency of the expression of Nanoluciferase when the two subunits of the AC are forced to get closer as well as the amplification process of the system.

These data suggest that the classical cytoplasmic BACTH system is functional in BTH101 strain and can discriminate the presence or absence of the target from a 90 min induction.

Membrane BACTH assay

To be able to test the membrane BACTH (mBACTH), OmpX 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 this azide-functionalized amino acid in the protein, which is able to fix a DIBO group - these modified proteins are called COMPs. 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 a DIBO group coupled with a fluorescent molecule : FITC. Results of the COMP, COMP-T18 and COMP-T25 proteins marked show a great protein expression on the external membrane. (See results here).

The mBACTH following results are obtained with 5µL of bacteria (24E+05 CFU).

Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the mBACTH generated with either
pOT18-Nlc and pOT25 : free sub-parts : negative condition,
or pOT18-Nlc-ZIP and pOT25-ZIP : Leucine Zipper : positive condition.
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.

With 1,48E+06 RLU of bioluminesce produced in the 0,5 mM IPTG condition compared to 9,02E+05 in the condition without IPTG and without ATP, it seems that IPTG increase slightly the transcription.
Additionally, with 2,55E+0,6 RLU of bioluminescence produced in the without ATP and 2mM ATP condition compared to 9,02E+05 in the without ATP and IPTG 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 don’t 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 from the 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.

Similarly to the BACTH system, 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.

Comparison of the efficiency of the classic cytoplasmic BACTH and the external membrane BACTH of iGEM Grenoble-Alpes team.

Comparison of the efficiency of the classic cytoplasmic BACTH and the external membrane BACTH of iGEM Grenoble-Alpes team.
Blank was done with 24E+05 CFU of untransformed BTH101 (RLU = 300) and subtracted from the measurements.

The results show a higher a quicker bioluminescence with the BACTH than with the mBACTH, this is due to multiple factors :
The proteins diffuse more easily in the cytoplasm than in the outer membrane and so both sub-parts are more likely to encounter each other in the cytoplasm than in the outer membrane. Whereof there is more functional adenylate cyclase in the cytoplasm than in the outer membrane.
With traditional BACTH, cAMP is produced in the cytoplasm making it directly accessible to the proteins that enable transcription (CAP), unlike mBACTH that produce cAMP inside the periplasm. Thereby, to be accessible by the CAP proteins cAMP need to diffuse in the cytoplasm thus increasing the time needed to enable translation and decreasing the quantity of cAMP reaching the CAP protein therefore reducing the amount of NanoLuc produced.

Nevertheless the mBACTH have a lower background noise thus allowing the discrimination of both condition. At the end the mBACTH is operational if the bioluminescence detection and quantification is well optimise for our system.

Conclusion

Biobricks BBa_K1638002 and BBa_K1638004 have been improved in order to allow a functional bacterial two-hybrid system in the bacterium periplasm.
The data show that OmpX protein is expressed at the external membrane of bacteria and that 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_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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