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RESULTS BIO
Main results :
Our project is based on aptamers which are single-stranded DNA able to recognize a specific target with a high affinity. In order to find potential aptamers, we choose 2 strains of bacteria, S. aureus and E. faecium and we performed 10 rounds of SELEX (Systematic Evolution of Ligands by EXponential enrichment) on both bacteria.
After these 10 rounds of SELEX, gel electrophoresis were made in order to verify that a high concentration of potential aptamers are still present.
Figure 1: Gel electrophoresis of 10 rounds of SELEX for E. faecium The gel is a 4% agarose one and the ladder used was a 50 bp ladder from Promega. The gel ran at 120 volts during 1 hour approximately.
We can observe that for the candidate aptamers selected against E. faecium we always had a single band throughout the rounds of SELEX (Figure 1). This gel demonstrates that a selection was applied on the initial DNA library because the bands are less brighter at the rounds 9 and 10 than the firsts rounds. This suggests that the most part of non-specific aptamers of E. faecium were removed during the washes made during the SELEX and that the specificity of the potential aptamers for E. faecium may have increased over the rounds.
Figure 2: Gel electrophoresis of 10 rounds of SELEX for S. aureus The gel is a 4% agarose one and the ladder used was a 50 bp ladder from Promega. The gel ran at 120 volts during 1 hour approximately.
As the previous one, the figure 2 demonstrates that the potential aptamers against S. aureus were selected because the bands loses their brightness over the rounds. However, on the round 9, we can notice 3 bands instead of one. It may be due to non specific amplification of DNA during the PCR. Despite this, it seems that these non specific amplifications were removed during the washes because they are not revealed during the round 10. So, we may think it will not interfere with the candidate aptamers.
Figure 3: Gel electrophoresis of the plasmid pET28:GFP at different step of the cloning experiment. The gel is a 1% agarose one and the ladder used was a 1 kbp ladder from Thermo Fischer. The gel ran at 120 volts during 1 hour approximately.
On the figure 3, we can observe the different steps of the cloning experiment. First of all, the plasmid pET28:GFP was digested using the restriction enzymes HindIII and BamHI. We can see a decrease on its size around 5 kbp which is a sign that the digestion went well. Then, the insert, either aptamers against S. aureus or those against E. faecium were ligated into the plasmid in order to create the biobrick BBa_K3176020. Thus, on the gel, we can see that after this step there is two bands for both samples, proving that the ligation was well performed.
Sequencing
10 rounds of SELEX were performed in order to select potential aptamer candidates. These aptamers were cloned into pET28:GFP in order to be sequenced as bacterial clones. A total of 15 bacterial clones for E. faecium and 39 for S. aureus were sequenced. Unfortunately, the majority of the sequences obtained did not correspond to an aptamer sequence. Only two bacterial clones contained the expected 40 nucleotides of our initial library. These two sequences were found for E. faecium strain, none was found for S.aureus. Here is an histogram showing the sequence for one of these two aptamers.
Figure 4: Sequencing profile of the aptamer selected against E. faecium The sequencing was performed by Eurofins genomics after we send them the transformed and isolated clone.
Characterization of BBa_K1073022
We chose to characterize BBa_K1073022, the eforRed chromoprotein, and focus on its fluorescence expression using two techniques: flow cytometry and fluorescence microscopy.
Flow cytometry
Throughout the flow cytometry, we would like to determine the transformation efficiency as well as the stability of the fluorescence expression. For that, four samples were analysed, a negative control containing non transformed E. coli, E. coli transformed and grew during 2 hours, E. coli transformed and grew during 12 hours and E. coli transformed and grew during 30 hours.
Figure 5: Flow cytometry of E. coli transformed with BBa_K1073022
On the left, the figure represents the number of bacteria which were analyzed.
On the top right, the figure represents the emission fluorescence of bacteria. Sample 3: E. coli transformed and grew during 30 hours, sample 2: E. coli transformed and grew during 12 hours, sample 1: E. coli transformed and grew during 2 hours, ctrl neg: negative control containing non transformed E. coli.
The sample 2 was our reference because the bacteria grew during 12 hours which is the appropriate time of an overnight culture. We observed that its fluorescent peak corresponds to around 103 and 104. Moreover, only 40% of bacteria were fluorescent. The sample 1 had a slight shift toward the left side but 50% of the bacteria population were fluorescent. Finally, the sample 3 emitted fluorescence in the same range as the sample 2 but fewer bacteria were expressing fluorescence.
Hence, due to the low number of bacteria expressing fluorescence only 2 hours of incubation, it seems that the transformation efficiency is low.
When the selective pressure was removed, the bacteria continued to lose expression of the chromoprotein as wild type bacteria out-competed those with the plasmid. Moreover, due to the fact that the expression decreases over time, we may conclude that along time the bacteria may lose their fluorescence properties. It can be due to the fact that bacteria express fluorescence less over time.
Fluorescent microscopy
For this experiment, the expression of eforRed was characterized. Three types of samples were analysed, a negative control containing non-transformed E. coli, E. coli transformed and grew during 12 hours and E. coli transformed and grew during 30 hours. As we can clearly see, transformed E. coli express red fluorescence when excited at 580 nm.
Figure 6: RFP fluorescence of the eforRed chromoprotein
(A) E. coli DH5α before being transformed. (B) E. coli DH5α after being transformed using BBa_K1073022. Scale bars represents 10µm.
However, when we merged brightfield and mRFP images, we observed that the fluorescence is not expressed by all of the bacteria. This result is consistent with the percentages established during the flow cytometry experiment.
Figure 7: RFP fluorescence efficiency
Chemistry
Cyclic voltammetry
If the aptamers were correctly bound to the electrodes, they should act as a protective layer around the electrodes. When they are not in contact with bacteria, a potential corresponding to PBS is therefore measured. When bacteria are added, the aptamers change conformation and allow the ions in solution easier access to the electrode: therefore, the overall potential increases.
Figure 8: Explanation of the increase of potential in presence of bacteria
Cyclic voltammetry is an analytical chemistry technique, which involves the use of a redox couple. A solution containing electrochemical species are deposited onto the electrode. Then, using a potentiostat, a potential is applied to the electrode in a sweep: starting from low potentials to higher ones, then back again. The response in current is measured and plotted against the potential.
Figure 9: Cyclic voltammetry ref: Edmund Dickinson, Modeling Electroanalysis: Cyclic Voltammetry, COMSOL Blog
The above graph is the usual waveform of a cyclic voltammetry analysis. When sweeping towards higher potentials, the electrochemical species are oxidized, which translates into a peek in current. When sweeping back, the oxidized species are reduced, theoretically yielding an equivalent peak, but in negative current. We conducted the experiments both with nanotubes-only electrodes, and with aptamer-functionalized electrodes. If we correctly deposited aptamers, they should form a protective layer around the electrode like mentioned previously: therefore, the electrochemical species should have a harder time getting close to the electrode to be oxidized and reduced, and the peaks should be lower in intensity.
While no difference was noted in the oxidation peak, it is quite clear that the reduction peak is more intense and slightly shifted towards the lower potentials. This modification occurs on all electrodes for all the strains: it was not predicted by the theory, which states that if the binding was correct, both the oxidation and the reduction peak should be less intense.
The only conclusion we may draw from this experiment is that we have indeed modified the electrodes, all in the same way, but we do not exactly know the state of the surface at this point.
The most likely explanation for this inconclusive experiment is that the fixation of the aptamers wasn’t complete. This might be explained by the fact that the drops of the fixating solution evaporated very quickly: some aptamers may therefore not have had time to bind correctly and instead adsorbed unto the nanotubes. The protective layer we had hoped to detect using cyclic voltammetry was therefore not thick enough for us to notice a difference.
Bacterial detection with aptamers/carbon-nanotube electrodes
Once the aptamers are attached to the carbon-nanotube electrodes, pathogens can be selectively detected by potential change. Using our arduino as a voltmeter, we have recorded the response profile of our electrodes following the addition of bacteria at different time points. Graphs represent potential (mV) on y-axis and time (s) on x-axis.
For each test, the same protocol was performed, as following :
50 µL of 1X PBS are added on the electrode.
After stabilization of the potential (2-3mins), 25 µL of bacterial
solution are deposited on the PBS droplet and mix up and down.
Change in potential is recorded.
After 100 seconds, 25 µL of bacterial solution are added once again.
Five conditions were tested.
As a negative control, PBS has been added on the surface of the electrode coated with our own aptamers designed for E. faecium: BBa_K3176020. On Figure 10, two peaks are observed perfectly corresponding to the moment when the drop is added. This environment disturbance induces a temporary change in potential, before quickly returning to the basic potential.
Figure 10: Negative control with PBS
We then detected a signal in the presence of the targeted bacteria E. faecium. Figure 11 shows a high change in potential with a staircase profile. Bacteria are well able to bind to the aptamers, inducing a change in their conformation.
Figure 11: E. faecium injected in a drop on an electrod with aptamers selected for E. faecium
We finally could observe that our electrodes coated with E. faecium aptamers are only specific to this strain. Figure 12 shows that a solution of E. coli does not induce any staircase potential jumps as observed for E. faecium. Indeed the profile of the graph is similar to the PBS control : with two peaks when the drop is added, before quickly returning to the basic potential. This observation proves that our aptamers designed for E. faecium are specific to this strain.
Figure 12: E. coli injected in a drop on an electrod with aptamers selected for E. faecium
Finally, E. faecium bacteria were added within a negative urine control (SurineTM), simulating analysis conditions. Once again, a staircase trend in the curve showed that bacteria are well able to bind to the aptamers.