Team:Grenoble-Alpes/Improve
Eindhoven-2015 iGEM project’s aim was to develop a “universal membrane sensor platform for biosensors”. This year, Team Grenoble-Alpes is designing a new tears biosensor system based on Eindhoven-2015 iGEM project. Both projects have a common base, the same receptors are used at the external surface of bacteria : clickable outer membrane protein called COMP. OmpX is an outer membrane protein with the C- and N-termini in the intracellular domain. To be able to use OmpX as a scaffold, a unnatural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups, this modified protein is called COMP - for Clickable Outer Membrane Protein (More information here). The complex aptamer fixed to a COMP is then called a COMB. Aptamers are clicked on COMPs by a copper free click chemistry using DIBO and are used to recognize target biomarkers in the external environment of the bacteria, thus bringing two COMBs closer and triggering a specific signalling cascade highlighting the specificity of this interaction. Gif 1: Animation showing the recognition of a specific biomarker with a specific aptamer fixed to COMPs proteins. T18 and T25 are the two subparts of the adenylate cyclase that enable the BACTH to work. The aptamer used can recognize two different epitopes of the same target thus increasing the probability of interaction between one T18 and one T25 subparts that couch one target. This particularity enhances the system specificity, because two epitopes of the target biomarker have to be recognized to induce the signalling cascade. In Eindhoven project, two different aptamers are used, each able to recognize one specific epitope (More information). Which means that two aptamers recognizing two different epitopes are necessarily needed in order to induce the signalling cascade, reducing the probability of interaction between one T18 and one T25 subparts (LINK aptamer princesse Pauline).
Considering the intracellular part of the system, both teams have differents ways to operate the signaling event in connection with a change in the proximity of the membrane protein. Eindhoven 2015 team chose to use the split luciferase system, and a Bioluminescence Resonance Energy Transfer (BRET) system by using the BRET pair NanoLuc and mNeonGreen proteins (More information here). With one biomarker detected, only one protein is activated thereby the signal might be very low if there are only a few biomarkers in the sample.
The Grenoble-Alpes team aims to develop an Outer-membrane Bacterial Adenylate Cyclase Two Hybrid system by using the adenylate cyclase from Bordetella pertussis. This Adenylate Cyclase (AC) has the particularity to be split into two subparts - T18 and T25 - unable to work unless they are physically close (More information: page BACTH site NeuroDrop). To perform a Bacterial Adenylate Cyclase Two Hybrid (BACTH), the two subparts of the AC are fused to the two proteins of interest analyzed, thus constituting two hybrids. The physical closeness of these hybrids induces their heterodimerization and results in a functional complementation between T25 and T18 fragments. Therefore, cAMP is produced, due to the recovery of the enzyme’s activity (More information). This system allows a reduction of potential false positives, because the heterodimerization of the two hybrids is reversible. This means that if the closeness of the hybrids is not stabilized by the target and is reached randomly due to the free movements of the proteins, the signaling cascade will not last in time. In this case, the two AC subparts are fused to the N-terminal ends of COMPs with a Gly-Gly-Ser Linker (GGS) of 54 amino acids, in order to ensure a sufficient flexibility. When COMPs and the aptamers catch the extracellular target, they get closer, thus allowing the reconstitution of a functional adenylate cyclase due to the physical proximity of the two subunits. (Gif 1). The enzyme is operational again and can produce the production of a high quantity of cAMP (around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute [1], the molecule responsible of the signal transduction in the bacterium. cAMP molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP). One cAMP molecule binds to one transcriptional activator CAP; then two cAMP/CAP complexes are needed to activate the expression of the lactose promoter. Because of the high quantity of cAMP diffusing in the cytoplasm of the bacterium [2], the reporter gene is continually activated as long as cAMP is produced (Gif 2) Gif 2: Animation highlighting the signalling cascade inside the bacteria. cAMP molecule, after its diffusion in the cytoplasm of the bacteria, is coupled to CAP in order to activate the promoter lactose plac. The high enzymatic activity [1] of Bordetella pertussis Adenylate Cyclase (AC) involves a high production of cAMP in presence of ATP in the bacterium (Figure 1) thus activating the signaling cascade with the CAP-cAMP dependant promoter. Hence this system is promising because it might have a great sensitivity and may give a great signal amplification for a low amount of target detected. Figure 1: Production of cAMP from ATP, catalyzed by the adenylate cyclase enzyme [4]. In contrast, when BRET is used - Eindhoven 2015 team project - the signal produced depends only on the quantity of target detected. There is no amplification, for one target detected only one protein will be activated and will generate fluorescence or luminescence thus limiting the detection. This can be a major limit if the target concentration is low !
The goal here is to prove that a membrane bacterial two hybrid (mBATCH) can be generated and can be functional by improving two biobricks from the original BACTH system:
In order to do this, 4 biobricks are created:
These two biobricks constitute the negative condition of the mBACTH (free sub-parts condition). OmpX proteins are fused to the AC subparts at their N-terminal ends, but they are not forced to get closer and move freely in the bacterial external membrane. 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.
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 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 [2]. This is a strategy to mimic the target recognition by the aptamers located at the bacterial cell surface. 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 (BBa_R0010). Two major factors affect this promoter :
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. Figure 2: Structure of the complete lac operon and effect of cAMP on CAP-dependent promoter. 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 (Figure 1). 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.
To compare the efficiency of the BACTH system created with the initial biobricks BBa_K1638004 (containing the T18 subpart) and BBa_K1638002 (containing the T25 subpart), quantification results in BTH101 strain are needed.
Those constructs will constitute the negative condition that will reveal the background noise of the initial BACTH system.
Those constructs will constitute the positive condition that will reveal how the signal increases when both sub-parts are brought together with the BACTH. Figure 3: 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 (Figure 4):
Those constructs will constitute the negative condition that will reveal the background noise of the initial mBACTH system.
Those constructs will constitute the positive condition that will reveal how the signal increases when both sub-parts are brought together with the mBACTH. Figure 4: Genetic constructions of pOT18-Nlc, pOT25, pOT18-Nlc-ZIP and pOT25-ZIP plasmids used to test the membrane BACTH in BTH101 strain. For the assay with the initial cytoplasmic BACTH, BTH101 are co-transformed either with pJT18 and pJT25-Nlc plasmids (negative condition), or pJT18-ZIP and pJT25-Nlc-ZIP plasmids (positive condition). For the assay with the membrane BACTH, BTH101 are co-transformed either with pOT18-Nlc and pOT25 plasmids (negative condition), or pOT18-Nlc-ZIP and pOT25-ZIP plasmids (positive condition). Then both cytoplasmic BACTH and mBACTH assays will be analysed and compared. To test the two different BACTH systems, 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 (Eindhoven-2015 iGEM project’s). 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 information) : “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 different experiments with 3 measurements per well are calculated. Data are expressed as the mean +/- standard deviation. Several controls are performed: The condition with 0,5 mM IPTG and 2 mM ATP is the experimental condition corresponding to the measure at 360 min. For the cytoplasmic BACTH, the following results are obtained with 5µL of bacteria (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. Table 1: Means of measurements obtained through 3 differents experiments with 3 measurements per well for each condition of the cytoplasmic BACTH generated either with pJT18 and pJT25-Nlc : free sub-parts (negative condition) or with pJT18-ZIP and pJT25-Nlc-ZIP : 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 strains, we measured 7.48E+06 RLU of bioluminescence produced in the 0.5 mM IPTG condition compared to 6.02E+06 in the without IPTG and 2 mM ATP condition, indicating that IPTG increase slightly the transcription. Additionally, with the same produced bioluminescence between the without IPTG and 2mM ATP and without IPTG and without ATP conditions, 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. Those observations give great clues on the way the system operates. Figure 5: 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 to the measurements. The Figure 5 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. To test the membrane BACTH (mBACTH), 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 which allows to click 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 Alexa 488 conjugated DIBO group. Fluorescent microscopy observations of the COMP, COMP-T18 and COMP-T25 clickable proteins show surface labelled bacteria indicating that the recombinant proteins are expressed at the external membrane of E.Coli. (Link vers les résultats). The mBACTH following results are obtained with 5µL of bacteria (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 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. Table 2: 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-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+06 RLU of bioluminescence produced in the without IPTG and 2 mM 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 helps the cAMP production by the periplasmic adenylate cyclase. Those observations give great clues on the way the system operates. Figure 6: 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 the measurements. 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 -. Similarly to the cytoplasmic BACTH system, 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. Figure 7: 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 to the measurements.
The results show a higher a quicker bioluminescence with the BACTH than with the mBACTH, this is due to multiple factors :
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. Biobricks BBa_K1638002 and BBa_K1638004 have been improved in order to allow a functional bacterial two-hybrid system in the bacterium periplasm by being fused with protein of the outer-membrane. The data show that COMP 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 two-hybrid can be successfully performed in the periplasm of bacteria which property is required for the sensing and detection of extracellular molecules.
By working for one year on this iGEM project, the team succeeded to prove that:
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.
Those three subparts of the project have been tested and approved separately. Further testing is possible by using:
that we also created but we did not had enough time to test their functionality. These biobricks are built and ready to be inserted into a plasmid containing the BBa_K3128001 (NanoLuciferase gene under cAMP inducible CAP dependant lactose promoter). Cloning of these three biobricks in a single plasmid is required, because a second compatible plasmid is needed to ensure the bacteria to have the molecular tools needed for the incorporation of the unnatural amino acid into the OmpX proteins. (cf click-chemistry parts). A co-transformation with these two plasmids have to be done in BTH101 strains, followed by a bioluminescence assay similar to the one that has been performed for the BACTH and mBACTH controls above, in order to confirm that the entire system is functioning. Then, a quantification of the smallest quantity of the biomarker - that can be detected with the system - will be required. This will allow to specify the sensitivity of our NeuroDrop system to detect biomarker in small volumes.
[1] Leusch, Paulaitis, Friedman. Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization. Am Soc Microbiol | Infect Immun. (1990) 
NeuroDrop
IMPROVE OF THE BACTERIAL ADENYLATE CYCLASE TWO HYBRID
BBa_K1638004: T18 domain of adenylate cyclase from Bordetella pertussis
BBa_K1638002: T25 domain of adenylate cyclase from Bordetella pertussis
Purpose
Materials and Methods
Bacterial Strain
Design of the plasmids for the classic BACTH assay
Design of the plasmids for the mBACTH assay
Transformations
Classic cytoplasmic BACTH and mBACTH assay
∅ 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
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.
Results
Cytoplasmic BACTH assay
* A T test was done for the values of time above 90 min and led to a p-value below 0.05.Membrane BACTH assay
* A T test was done for the values of time above 90 min and led to a p-value below 0.01.
* A T test was done for the values of time above 210 min and led to a p-value below 0.05.Comparison of the cytoplasmic BACTH and the membrane BACTH
Conclusion
Further testing
References
[2] Hantke, Winkler, Schultz. Escherichia coli exports cyclic AMP via TolC. J Bacteriol. (2011)
[3] Bornberg-Bauer, Rivals, Vingron. Computational approaches to identify leucine zippers. Nucleic Acids Research, Volume 26, Issue 11, Pages 2740–2746.(1998)
[4] Picture of the reaction ATP-cAMP. Khan Academy Website. Retrieved October 10, 2019, from https://www.khanacademy.org