Overview of Engineering

The essential part of our project is the production of a quantitative rapid test against the skin microbial community. For our test we focus on two parts. First, we targeted the dominant species of C. acnes and all gram-positive skin bacteria by using SELEX and aptamer technology. Secondly, we created a colorimetric detection method using PCDA polymer to observe binding of the aptamer to the ligand.

Here we present the engineering processes and how we tailored the engineering design cycle for the various experimental challenges in our project: MIKROSKIN.

Aptamer Production

Research

Aptamers are small, single-stranded oligonucleotides that can assemble to form complex structures. These folded three-dimensional structures form interactions with small molecule targets which can be detected, as shown in Figure 1. The aptamer libraries are obtained using the Systematic Evolution of Ligands by Exponential enrichment (SELEX) technology. SELEX is a basic technique for the selection of aptamers in vitro against various targets, ranging from small molecules like ATP to proteins to even whole cells. The interactions between the aptamer and its target involves different intermolecular interactions including van der Waal's forces, electrostatic interactions between charged groups and three-dimensional shaping by hydrogen bonds.

Due to the ability of aptamers to recognize and bind specifically to targets, they are often compared to antibodies, but the former can be crafted to bind to multiple different targets unlike antibodies which can only bind to immunogenic toxic targets. Aptamers are much smaller than antibodies, so they are able to fit into clefts and gaps on the surface of much larger target molecules that antibodies sometimes cannot reach. They are also more resilient against heat and organic solvents. These properties provide a huge market for aptamers to be used in, e.g, the field of diagnostics for targeting biomarkers, such as cancer, and in the drug delivery process.

Figure 1: What are aptamers? The structure was generated in BioRender using reference

Design

In our project, we aimed to develop two aptamers: one is targeting lipoteichoic acid (i.e. LTA), which is present on the cell wall of all gram-positive bacteria. As they are dominant on the skin, teichoic acid can be used for quantification of the skin microbiota. The second is an aptamer targeting a strain of C. acnes, which is dominant in acne-prone skin. The two SELEX libraries (for C. acnes surface and LTA) are incubated with their respective targets, resulting in the binding of some aptamers to the targets and the unbound sequences being removed by washing. The bound sequences are eluted from their target by heat and then amplified by error-prone PCR. This concludes the first round of SELEX, but in order to obtain the desired aptamers with high binding affinity, multiple SELEX rounds are necessary. SELEX is therefore a process of directed and targeted in vitro evolution.

Building and Testing

In the lab, we have created our own protocol for the production of the two aptamers. We have consulted with our instructor, Dimitri Van Simaeys, and referred to research papers to find the optimal protocols for aptamer selection. We have faced certain challenges while designing the aptamers, such as ordering the wrong primers and receiving undesirable PCR results for two months. We have hypothesized and tested several protocols over the months in the lab; when we observed primer dimer formation in the PCR products, we altered the annealing temperature from 55 degrees Celsius to 57.5 degrees Celsius, but when it still was not working, we decided to change the concentrations of the PCR components, closely following the protocols by Takara HS Taq. We also faced other setbacks like contaminated PCR components, low library amplification, and having amplification in the non-template control.

After several trials and optimizations of the protocols, we finally succeeded with the amplification of the aptamer library! We are currently troubleshooting the desalting step using GE healthcare Cytiva illustra NAP column NAP-5, after which flow cytometry is performed to conclude the first SELEX round.

Learning and Improving

From our journey in the lab, we have learned a lot about troubleshooting techniques when it comes to PCR reactions; we emerged victorious towards the end. Although we did not have enough time to produce our aptamers, in the end, we improved our techniques so that future iGEM teams working with aptamers will be greatly benefiting from them. We have shared information about our journey in the troubleshooting guides, including detailed procedures and tips and tricks to keep in mind when working with aptamers in the laboratory.

• In vitro SELEX Troubleshooting Guide
• Cell SELEX Troubleshooting Guide

Detection using PCDA

Research

After researching ways to create the quantitative test to examine the dysbiosis on the skin, we decided to base our detection tool on a colorimetric method in order to improve the visibility of the results for the naked eye and spectrophotometers. This was achieved by using the colour changing property of polydiacetylene (PCDA) polymers when exposed to stressful conditions, such as chemical, mechanical, heat, or radiation.

Design

The premise of this idea is that a stress-induced structural change occurs, regardless of the target being lipoteichoic acid or a C. acnes cell. It is visualized by a colour change from blue (monomeric solution) to red (polymerized) (Figure 3), proportional to the amount of bound target. We can then detect this by measuring the absorbance using a spectrophotometer, and thereby use it as a semi-quantification test of dysbiosis.

Figure 2: Polymerization of crystalline diacetylene monomers under heat stress. The structure was generated in ChemDraw using reference

Building and Testing

We initially tested this detection method by inducing UV radiation overnight to the PCDA polymeric solution and observing a colour change. However, the colour change was very subtle (Figure 3).

We then moved on to assessing the colour change by inducing heat stress using a Bunsen burner for a few seconds. In this case, the colour change was much faster and we could see the blue solution turning into a bright orange (Figure 3). Thus, we concluded that the heat stress colour change is easily detectable.

Figure 3: PCDA solutions in different concentrations, when subjected to some stress

Learning and Improving

From this improvement in the operationalization technique, we found a better detection approach. We think it might be because there is a higher energy flux transfer when heating for a few seconds rather than placing the polymeric solution under UV radiation overnight. We can also use this rapid detection test for any new developing aptamer, so it can be used as a high throughput test depending on the number of targets.

References

• https://doi.org/10.1038/nrd3141

• https://doi.org/10.1007/s40257-020-00531-1

• 10.1038/mtna.2014.32

• 10.1016/j.forsciint.2011.09.019

• https://doi.org/10.1002/app.33250