PCDA Synthesis and Polymerisation

Our detection method is based on the 10,12-Pentacosadiynoic acid (PCDA) polymer vesicles which can change colour when subjected to various kinds of stress, such as chemical, mechanical, heat and radiation.

The first step was to synthesise PCDA polymers from crystalline PCDA monomers. In their polymer form, PCDAs adopt vesicle formations in a water solution. This solution can then be stored at 4 degrees Celsius for extended periods of time.

We tested the PCDA solution for reactivity to heat and UV-radiation. The results of this test are shwon in Figure 1. For heat, a few mL of the solution was poured into a glass test tube and subjected to a burner flame. After a few seconds, the solution turned to a bright orange, indicating a structural change. When it comes to UV-radiation, the colour change is a lot more subtle, changing to a deep purple when left overnight under UV-radiation. This is probably due to the UV lamps' supplied energy being relatively low, especially compared to the burner's energy flux into the solution.

Figure 1: PCDA solutions subjected to different stress conditions (heat, UV radiation, normal), and subsequently diluted for spectrophotometric measurements.

Spectrophotometric measurements of the colour change were performed with the solutions from Figure 1, and the results are shown in Figure 2. The absorbance maxima changes from around 640 nm to around 500 nm. Both peaks visible on the spectrum are shifted to lower wavelengths when the PCDA solution is heated.

Figure 2: Absorbance spectrums, shown with different coloured curves of A: Blue PCDA at 1 mM (Black), 0,1 mM (Red) and 0,01 mm (Blue); B: Red PCDA at 1 mM (Blue), 0,01 mM (Purple), 0,01 mM (Red) and 0,001 mM (Black).

PCDA - Protein A Aptamer Conjugation

The next step was to conjugate our PCDA with the aptamer and then test it using protein A. After incubation of the protein A with the PCDA-aptamer conjugate we did not detect any colour change so we needed to test if the protein A aptamer is actually binding protein A. We investigated the protein A aptamer binding affinity with three different experiments: first, using protein A-coated beads, then with chromatography, and finally by performing a binding assay.

Protein A coated beads

Early on, after noticing that the protein A aptamer might not bind to its target, we used protein A coated beads for testing the binding qualitatively. After following the protocol provided with the beads, we ran the fraction from elution in an agarose gel, which is shown in Figure 3. After elution, the one sample of the fraction was desalted, in order to have a less harsh environment for the aptamers. However, as can be seen on the gel, the wells appear empty, which means that the aptamers most likely did not bind to protein A. It is nonetheless possible for the binding to be too weak, resulting in very little aptamers being eluted and thus not showing up on the gel.

Figure 3: Agarose gel with the protein A beads products. In the last column had the product after the elution of the beads where the eluted aptamers are supposed to be. The second-to-last column had the eluted product which was also subsequently desalted.

Chromatography using a protein A coated column

Since the test using the magnetic beads could have a too low concentration in the elution for it to be detectable on a gel, we set up a chromatography experiment with a protein A column. The resulting chromatogram is shown in Figure 4. Fractions from the sample application run-through and from the elution were analysed using an agarose gel, which is also shown in figure 4. The wells containing the sample application run-through had a lot of contents, while the fractions from the elution were empty. This again shows that the protein A aptamer was not binding to protein A.

Figure 4: Chromatography results. The chromatogram is shown at the top. Tubes 1 to 4 are from sample application while tubes from 15 to 24 are fractions from elution. The agarose gel with the chromatography products is shown at the bottom. Each well is labelled with the corresponding tube from the chromatography. From left to right, the first well has ladder, the next four have samples from tubes 1-4 and the last ten have samples from tubes 15-24.

Binding assay using a fluorescent protein A aptamer

The previous results suggest that our protein A aptamer is not binding protein A as expected. In order to finally figure out the aptamer affinity issues we designed the following experiment using protein A aptamer labelled with the fluorophore fluorescein (5' 6-FAM aptamer).

We incubated 100pmol, 200pmol and 300pmol of fluorescent aptamer in triplicates in a 96-well plate coated with protein A for 45 minutes. Then, washed the wells with PBS to remove unbound aptamer molecules and measured the fluorescence intensity in a plate reader.

To infer the pmol of aptamer that had successfully bound to protein A, we created the following standard curve using known pmol quantities of 5' 6-FAM aptamer.

Figure 5: 5' 6-FAM aptamer standard curve showing the relationship between RFUs (Relative fluorescence units) and aptamer amount (pmol). A linear regression model was used to determine the line, regression equation and R^2 coefficient.

Using the regression equation, we inferred the pmol of aptamer that bound to the protein A.

The total percentage of bound aptamer was then calculated for each sample by dividing the pmol of aptamer bound by total pmol aptamer incubated.

Table 1. Amount of aptamer bound (pmol) and percentage of bound aptamer determined by the binding affinity assay.

Based on these results, we concluded that the protein A aptamer we chose for our proof of concept does not have sufficient binding affinity to its target.

This conclusion is also supported by protein A - aptamer docking model (link to model page), where no evidence of obvious strong interactions was found.

SELEX

Another crucial part of our project was to create the two aptamers, one for lipoteichoic acid and one for C. acnes, using SELEX. We faced a lot of problems during this process. The main obstacle was the optimization of the PCR. From wrong concentrations of PCR reagents to contaminations and wrong primers we spent a lot of time troubleshooting in order to make the PCR work since it is the most important part of the SELEX process. One example is shown in figure 6, which displays an agarose gel with PCR troubleshooting products, where two different polymerases, temperatures and primer concentrations were tried. Everything is written in detail in the engineering and in our troubleshooting guide.

Figure 6: Troubleshooting example. PCR products run in a 2% agarose gel. Two different polymerases (Takara Titanium Taq DNA Polymerase and DreamTaq DNA Polymerase), primer concentrations (0.5 mM and 1mM) and annealing temperatures (55°C and 60°C) were tested. Additionally, a non template control (NTC) for each polymerase and annealing temperature was run.

After all the hard work over the summer, we were delighted to get positive SELEX results during our last few days in the lab. We performed a successful first amplification of the libraries in order to figure out the right amount of PCR amplification cycles needed for the second amplification, based on the gel image shown in figure 7. Then the remainder of both libraries were amplified with 45 cycles of PCR in total, since the pre-amplification accounted for 15 cycles and the optimal number of cycles after pre-amplification was chosen to be 30.

Figure 7: Agarose gel showing library amplification PCR products, with LTA SELEX in the left part of the gel and Cell SELEX on the right part of the gel. For each, the wells have samples with an increasing number of PCR amplification cycles, from left to right. The strongest bands with the least amount of laddering were picked for both types of SELEX, and both corresponded to 30 cycles (highlighted with dashed boxes).

The PCR products from the second amplification can be seen in figure 8. Then, the PCR products were pooled for each SELEX type, after which the sense and antisense strands were separated. The sense strands were eluted and desalted, which yielded very little product as can be seen in figure 9, and we are unsure whether it is the right product that was obtained.

Figure 8: 2% agarose gel of the amplification products for (A): LTA SELEX and (B): Cell SELEX; after 45 cycles. For each type of SELEX, each well had a sample from a separate PCR tube. The last wells had non-amplified libraries as samples.

Figure 9: Agarose gel of fractions from desalting after the elution of the sense DNA strands. The first well from the left after the ladder had sample from the run-through of the sample, the next well had sample from the eluted fraction, and the second-to-last well had sample from the left-over volume that came after the sample elution.

As SELEX is a repetitive process, we were able to prove that each part of our protocol works. Given more time, we would have worked towards achieving multiple successful rounds of SELEX.

BioBrick Engineering

We set out to create a recombinant protein A labelled with the yellow chromoprotein amilGFP as a reporter. In order to do this, we selected using pre-existing parts from the registry: the amilGFP BioBrick (BBa_K1073024), as well as staphylococcal protein A BioBrick (BBa_K103003).

Following our cloning strategy (Experiments > BioBrick engineering), we first successfully linearised the plasmid containing the amilGFP BioBrick by digesting with the SpeI and PstI restriction enzymes.

Figure 10: Digested and undigested product in 1% agarose gel. Lane C shows the undigested plasmid. Two different bands can be observed, corresponding to two different conformations of the DNA plasmid: open coiled (OC) and Covalently Closed Circle (CCC). Lane D shows the digested plasmid product.

Once we confirmed the plasmid had been linearised, we isolated it by performing gel extraction.

Figure 11: 1% agarose gel with linearised amilGFP plasmid, before (A) and after (B) the gel extraction. (A) AmilGFP plasmid was digested with SpeI and PstI. (B) Using the UV table, the fragment of interest was isolated, leaving out the smear.

Then, we moved on to amplify the protein A BioBrick by PCR, using VF2 and VR standard primers. The PCR product had the expected size: ~ 500 bp.

Figure 12: PCR amplification products on a 2% agarose gel. Lanes 1 to 3 contain the same PCR product. Lane 4 is PCR negative control (non-template control).

After performing a PCR clean up, the amplified protein A BioBrick was digested using XbaI and PstI restriction enzymes. The expected fragments were:

• Protein A BioBrick -> ~ 235 bp.
• VF2 - XbaI -> 133 bp
• PstI - VR -> 154 bp

We could not use PCR clean up to get rid of the digestion leftovers, since it removes oligos up until 40 bp. Therefore, the protein A BioBrick (digestion product) was isolated by gel extraction.

Figure 13: 1% agarose gel with digested protein A BioBrick, before (A) and after (B) the gel extraction. (A) The electrophoresis was run until the protein A BioBrick (~235 bp) was separated from other digestion products (~150 bp). (B) Using the UV table, the fragment of interest was isolated successfully.

The next step was to ligate the protein A BioBrick and the vector containing amilGFP plasmid to create the recombinant protein. Compatibility between XbaI - SpeI and PstI - PstI sticky ends allowed for directional cloning of the protein A BioBrick into the amilGFP plasmid. Ligation was performed by incubation of both parts with T4 ligase. The ligation product was then used to transform TOP10 E. coli competent cells.

Several yellow colonies, indicating amilGFP expression, were selected to set O/N cultures, from which glycerol stocks and minipreps were prepared. Sanger sequencing confirmed our plasmid had the desired sequence.

Figure 14: Yellow E. coli colonies due to amilGFP expression on LB-agar plate.

Ultimately, in order to create a functional recombinant protein, we had to mutate two sequential stop codons at the end of the amilGFP coding sequence by using Quikchange site-directed mutagenesis. During the PCR step, TAA STOP codons were substituted by TCA Serine codons thanks to the primers, which had these modifications incorporated (Figure quikchange). Three different annealing temperatures were used in the PCR: 55 degrees Celsius, 58 degrees Celsius and 60 degrees Celsius.

Then, DpnI endonuclease digested the non-mutated parental DNA strands, and the final product was transformed into TOP10 E. coli competent cells.

Figure 15: Quikchange Forward and Reverse primers are complementary to the target sequence at the end of the amilGFP CDS (BBa_K1073024), but include the desired point mutations (mismatches) that were incorporated by PCR. Retrieved from SnapGene.

Plates transformed with products from 55 degrees Celsius and 58 degrees Celsius PCR annealing temperatures had colonies. Expression of functional amilGFP could be confirmed with the presence of bright yellow colonies in LB-agar plates and yellow cell pellets in liquid culture.

Finally, the sequence of the newly engineered amilGFP-proteinA BioBrick (BBa_K4071000) was verified by Sanger sequencing and the part was validated.

Figure 16: Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa_K4071000).