Team:Grenoble-Alpes/Results

NeuroDrop

Results

Results of the Aptamer part

The aim of these experiments was to prove the recognition of the target by specific aptamers, select two of them and then check the potential competition between the two. These chosen aptamers are intended to be clicked on the mutant OmpX protein and by recognizing the target to bring together the T18 and T25 subparts bound to the OmpX proteins.

The first step was to characterise them one by one to prove the recognition and then, secondly to design a “sandwich” system. That means finding two compatible aptamers, which are not in competition one with the other. That is to say they have to recognise two different recognition sites, or a large enough site which can link two folded sequences.


The technique we used is the fluorescence polarization (see protocol). This permits to determine a fluorescence anisotropy, which fluctuate according to the concentration of the target and its recognition by the aptamer.
Fluorescence anisotropy measures the rotational mobility of the fluorophores that are excited with polarized light. Small molecule fluorophores undergo rapid rotation, thus depolarizing the polarized light during emission.


A fluorescent molecule has a bigger anisotropy if it is cluttered, because it is less movable. That is why we should observe an increase of the anisotropy in function of the increase of the target concentration. If the aptamer recognises the target, it will be bigger and its anisotropy will increase linearly until saturation.


With two aptamers, we can suppose two different cases to prove the absence of competition. We can have either an anisotropy gain compared to the case with one aptamer or any modification of the anisotropy. If a competition exists between the two aptamers, we should observe a decrease of the anisotropy.


We first started by working with aptamers of alpha synuclein described by Kaori Tsukakoshi et al. [1] . However, we have not been able to characterize them with fluorescence polarization. In addition, the method described in the publication, ELONA, was far too expensive for us to test. In addition, the described recognition time of three days was not adapted to our objective of rapid detection.


We finally studied on aptamers of Tau protein, also involved in neurodegenerative diseases.The three different aptamers we have tested come from the publication of Samuele Lisia et al. [2] The protein isomer we used as target was hTau40. hTau40 is the longest isoform in the human central nervous system with its 441 residues. It is also called Tau 441 for this reason.It contains the entire sequence of the Tau protein including both alternatively spliced N-terminal exons. Thus it possesses the two hexapeptides that are essential for aggregation of tau into paired helical filaments (PHFs) involved in the Alzheimer disease [3]


We chose them in function of their specificity and the theoretical compatibility of their sequence by two-to-one comparisons.To do this, we have looked at the possible secondary structures of oligonucleotides as well as their hybridization with the M Fold and Pearl Primer software.
Here are the chosen sequences below:

For each test we made a range with a fluorescent aptamer in the well in presence or not of a second non fluorescent aptamer. The tests have been realised in duplicate. The plate was read instantly after the aptamer was added to the target. Here are the results obtained:

Figure 0: Measurement of the anisotropy using fluorescence polarization. in blue the Ranges one of a fluorescent aptamer (blue) and ranges oh one fluorescent aptamer mixed with a non-fluorescent one (red). (1) (2) (3) 3146F is the fluorescent aptamer, 3146, 433 and 4133 are not labelled. (4) 4133F is the fluorescent aptamer, 433 is not labelled.

First of all, there is a linear increase in anisotropy as a function of Tau protein concentration which reaches a plateau at high concentrations (1.48 µM) suggesting a saturation phenomenon.(Figure 0)

For the four different tests, we do not have observed any anisotropy gain. Considering the standard deviation, we cannot conclude that there is a real loss of anisotropy due to competition between aptamers. We can only assume that the anisotropy appears constant.

Therefore, according to the hypotheses previously stated, we can conclude from the results that the aptamers recognize the target instantly. In addition, there is no competition among the different selected aptamers. In the case of 3146F and 3146, it suggests that this aptamer potentially recognizes two different sites on the target.

Nevertheless, one couple of aptamers seems to be more appropriate in the context of the project: namely the 3146/3146. Using only one aptamer to click in our system presents a considerable advantage. Indeed, using two different aptamers 1 and 2, the probability to click the first one on the OmpX-T18 complex and the second one on OmpX-T25 leading to the complexes 1-OmpX-T18 and 2-OmpX-T25 is rather weak. In contrast, using a single aptamer able to recognise two different sites triggers better probability to activate the system provided that the T18 and T25 subparts are next to each other.

Results of the Click-chemistry part

The purpose of these experiments was to confirm whether 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 in 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 15hours at 18°C.

Bacteria were incubated with 10mM of Click-iT ™ Alexa Fluor ™ 488 sDIBO Alkyne for 10 minutes.

I - Negative Controls

A - 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 an 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 spontaneoulsy 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 labeling of BL21 E.coli cultured in presence of pAzF.

B - BL21 Ecoli + pEVOL-pAzF + pAzF

In this experiment, we wanted to check if the presence of 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 does not 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.

The data presented in Figure 1 and Figure 2 are control conditions showing that:

  • Alexia Fluor 488 conjugated DIBO does not click on proteins that have not incorporated the pAzF.
  • Alexia Fluor 488 conjugated DIBO does not diffuse and accumulate into the cytoplasm.

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

II - Positive Controls

A - BL21 E.coli + pEVOL-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 COMP protein 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 labeling of BL21 E.coli containing pEVOL-pAzF and COMP cultured in presence of pAzF.

B - BL21 E.coli + pEVOL-pAzF + pAzF + COMB-T18 or COMB-T25

COMPs either fused to the T18 sub-part of adenylate cyclase or the T25 sub-part, were expressed in the presence of pAzF 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 releaving 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 OmpX.

Figure 4: Fluorescent-conjugated DIBO labeling:
(A) BL21 E.coli containing pEVOL-pAzF and COMB-T18 cultured in presence of pAzF.
(B) BL21 E.coli containing pEVOL-pAzF and COMB-T25 cultured in presence of pAzF.

Conclusion

These data establish the proof of concept of our biological system.

Culturing recombinant bacteria transformed with pEVOL-pAzF and a plasmid containing 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.

Results of the outermembrane Bacterial Adenylate Cyclase Two Hybrid Assay (mBACTH)

The following parts parts were use to characterise the outer-Membrane Bactereia Adenylate Cyclase Two-Hybrid aspect of this part.
BBa_K3128017 and BBa_K3128018 correspond to the negative condition.
BBa_K3128026 and BBa_K3128027 correspond to the positive condition.

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:

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.

mBACTH_plamides
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.

Results

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 cf Team Eindhoven 2015

The low temperature allows a native protein folding and membrane insertion to avoid 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:

“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 the 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.

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 different 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 measurement.

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 increases 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 has 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 are 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.

Nanoluciferase Visual Luminescence

Finally, here is a picture with 48.10E+06 bacteria to see a visual difference in the bioluminescence intensity with and without leucine zipper.

In 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 sensing and detection of extracellular molecules.

Results of the reporter gene

Our detection system is based on the use of a BACTH. The point is to conditionally induce the expression of the gene upon interaction of the two sub-parts of Adenylate Cyclase (AC) are physically close, which only occurs when the target is present in the sample and detected by our NanoDrop system.

The re-constitution of AC then enables cAMP production, which will activate a CAP dependent promoter allowing the transcription of the downstream gene.

In order to perform the assay we needed to use an AC deficient bacteria strain (BTH101) that do not produce endogenic cAMP. This property prevents any transcription from CAP dependent promoter such as lactose promoter.

For the choice of the promoter, we decided to use the lactose promoter (a CAP dependent promoter) and we have demonstrated its repression in the absence of cAMP (in the AC deficient bacterial strain), thus preventing any transcription of the following gene : the reporter one (see table and figure 1).

To resume, the expressed/overexpressed of the gene is under the control of cAMP. For a good sensitivity of our system, statistically different signals have to be recorded on negative sample (no AC reconstitution) and positive sample (constitutive reconstitution of sub-parts of the AC).

To prove that the reporter gene efficiently works in our system different conditions were tested.

First the leak of our reporter in the absence of cAMP was measured by transforming the plasmid containing the BioBrick PLac_NanoLuc in BTH101.

Then the free sub-parts condition was tested by co-transforming two plasmids in BTH101: pUT18 containing the AC sub-part T18 and pKT25_NLuc containing both the AC sub-part T25 and the BioBrick PLac_NanoLuc.

At condition imitating the target's recognition was tested.

Leucine-Zipper (LZ) were used to mimic the presence of the target and the physical connexion between both sub-parts. LZ have the capacity to form homodimer and so were added at the end of both sub-parts making them able to stick to each other in the absence of target thus restoring the AC activity.

Two plasmids were co-transformed in BTH101: pUT18-LZ containing the AC sub-part T18 fused with an LZ at the C terminal and pKT25-LZ_NLuc containing both the AC sub-part T25 fused with an LZ at the C terminal and the BioBrick PLac_NanoLuc.

If there is a significant difference of luminescence between the free sub-parts condition and the target detection imitating condition then it will demonstrate that our reporter gene is working in our system.

It will also show the cAMP dependent on/off transcription switch.

The assay

Bacterial cultures were induced with 0.5 mM of IPTG at an Optical Density of 0.6.

The subtract for Nano Luciferase (furimazine) was added as follows : for 50uL of bacterial culture in a well, 49uL of NanoGlo Assay Buffer and 1uL of NanoGlo Assay Substrate were added.

The bioluminescence expressed in Relative Luminescence Units (RLU) were recorded in a black NUNC 96 wells plate.

Two different bacterial cultures (sample) were assessed per experiment in duplicate (except for the 24 hours condition).

Blank was done with non-transformed BTH101 (RLU = 300) and subtracted to each measurement.

The reults

The second well for sample 2 was removed because the substrate was omitted.

Figure 1 : Bioluminescence means (unit: RLU) in BTH101 containing the NanoLuciferase gene without endogenous adenylate cyclase (grey) in BTH101.

Bioluminescence was recorded at different time points post induction.


Figure 2 : Bioluminescence means (unit: RLU) in BTH101 containing the pUT18 and pKT25_NLuc plasmids (orange) in BTH101.

Bioluminescence was recorded at different time points post induction.

Figure 3 : Bioluminescence means (unit: RLU) in BTH101 containing the pUT18-LZ and pKT25-LZ_NLuc plasmids (blue) in BTH101.

Bioluminescence was recorded at different time points post induction.

Conclusion

Figure 4 : Recombinant bacteria containing the NanoLuciferase gene without (grey) or with the pUT18 and pKT25_Nluc plasmids (orange) or with the pUT18-LZ and T25-LZ_Nluc AC sub-parts were culture. Bioluminescence was recorded at different time points post induction.

Those measurements highlight two major points:

First, the luminescence produced by the strain without AC (grey) is low and stable over time.

It can be considered as the background of the system.

In contrast the Bioluminescence recorded in the recombinant bacteria containing the free sub-parts of AC (orange) and LZ mediated reconstituted.

AC (Blue) showed a 1 to 10 order of magnitude increase and evolved over time to reach 5.9 E+ 07 +/- 4.9E+06 and 1.6 E+08 +/- 1.5 E+06 respectively 24 hours after induction.

These data suggest that T18 and T25 randomly reconstitute in the cytoplasm leading to cAMP production and expression of the NanoLuc reporter gene.

However, LZ mediated interaction of the two sub-parts of AC leads to a significant* increase of NanoLuciferase overexpression when compared with the random reconstitution.

* A T test was done and led to a p-value lower than 0.05.

Thanks to this BioBrick it is then possible to measure the difference between both conditions: random or LZ mediated reconstitution of AC.

As a consequence, this system could be adapted to our NeuroDrop project for the detection of extracellular biomolecule through the biosensor system conjugated to aptamers described elsewhere.

Software

To best prove that the software works, we used it to create protocols. The largest protocol we created with this software was a 10h-long protocol meant to measure the evolution of the luminescence of a solution through time. This protocol took us 1 minutes to define using the software. Here is the video of how we did it:

As we can see, the software perfectly performs its role:

  • It is simple to use
  • It allows to define protocols very fast
  • It allows us to save the protocol

The software itself can be reused also as a basis for a more complex device, or can be adapted to fit the evolution of the device.

Motors

To best prove that the motors work, here is a video of a motor moving. It might not be very clear at first sight that the movement is precisely controled, but the video was taken after the repetition of the same movement a 100 times. The result from that test is that after those 100 cycles, and without any control over the position other than telling the motor how many steps it had to make, we weren't able to detect an error in its movement.

EWOD

To show that we could use EWOD in our device, we decided to use a droplet of demineralized watter. This result confirms that this technology works as demineralized water has a lower concentration of ions compared to physiologic solutions. This means that if we manage to move a droplet of water using this technology, we will be able to move any solution we might encounter, as having an ionic solution increases the efficiency of the technology.

Measure of luminescence

To prove that our system can measure luminescence and return results that can then be used for diagnosis, we used a LED that we connected to a voltage generator. Then we tested the sensor for different voltages applied to the LED. Here is what we measured:

This would be useless if we couldn't relate this value to the bioluminescence of our solutions. We did that by comparing the luminosity of the LED to the one of the bioluminescent solutions, and even without using any device, we saw that the luminescence of the solution we got was higher than the light emitted by the LED. This proved that the system we developped to measure the luminescence is adapted.

A detail is also important in this curve: we have a precision of 0.07mA in our reading. This precision does take into account the noise. We are therefore extremely precise both on the measure and the conversion of the resulting current.