Team:Pasteur Paris/Results
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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
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.
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.
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
As a proof of concept, we used our arduino as a
voltmeter, to record the response profile of our electrodes following
the addition of bacteria at different time points. The objective was
to demonstrate the sensitivity and specificity of our own aptamers
designed for E. faecium.
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 was added on the surface of the electrode, following the previous protocol. 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.
Regarding the bacterial solutions, E. faecium was first added, following the previous protocol. The staircase trend in the curve shows that bacteria are well able to bind to the aptamers, inducing a change in their conformation.
E. faecium bacteria were added within a negative urine control (SurineTM), simulating analysis conditions. Once again, the staircase trend in the curve shows that bacteria are well able to bind to the aptamers.