Overview
The goal of this year's iGEM Stockholm team was to create a detection method based on aptamers bound to PCDA vesicles. When the aptamers bind to their target due to mechanical stress the PCDA will change colour indicating that the target is present. Quantification of the intensity of the colour change is proportional to the concentration of the bacteria present in the skin.
PCDA Polymerisation
Polydiacetylenes (PCDA) are a family of polymers created by the polymerization of diacetylene creating vesicles or tube structures.
(Reppy & Pindzola, 2007). They were used as a detection method due to their unique chromatic properties. 10,12-Pentacosadiynoic acid (PCDA) belongs in the family of polydiacetylenes and yields a blue colour in its primary form. Under the exposure of heat (thermochromism), mechanical stress (mechanochromism) or solvent (solvatochromism) it however changes colour from blue to red (Lebegue et al., 2018). For our experiments mechanochromism was of great importance, because, as mentioned before, binding of the aptamer causes mechanical stress - resulting in a colour change.
Formation of PCDA vesicles
In order to create the PCDA vesicles, we first dissolved the PCDA in chloroform, which we then we evaporated using gentle N2 flow. Following this, we dissolved the PCDA in water using sonication. The end product was an emulsion with white colour. The next step was exposure to UV light to induce polymerization which in turn results in a colour change from white to blue. This protocol has been used before for the creation of PCDA vesicles (JT et al., 2016; Wu et al., 2012). The solution was stored at 4 degrees Celcius until further use.
Figure 1: PCDA Vescicle Formation
Conjugation of the PCDA vesicles to the aptamers
The conjugation of the PCDA vesicles to the aptamers is based on the Carbodiimide method. This method is used for the creation of amide bonds between carboxylates and amines by adding dicyclohexylcarbodiimide (DCC). DCC is a crosslinker that works by creating a reactive intermediate that reacts with nucleophiles like amines resulting in an amide bond (Shah & Misra, 2011)(Hermanson, 2013). In our experiments EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) was used. EDC is the most popular carbodiimide for conjugating carboxylates with amines and it is used together with NHS (N-hydroxysulfosuccinimide) in order to stabilize the reactive intermediate.
We deemed this method suitable for our purposes, as the PCDA has carboxyl groups and the aptamer is a small DNA sequence with amine groups. The protocol we followed was based on a paper by Wu et al., (2012).
Figure 2: Biosensor mode of action
Bacteria Cultivation
Another important part of the project was the cloning of a biobrick, which was made by assembling two existing parts from the registry: part BBa_K103003 coding for staphylococcal protein A, and part BBa_K1073024 amilGFP. Firstly, the two biobricks had to be expressed and harvested through PCR and gel extraction. After that, the two parts were ligated and assembled into a vector. In order to have a functioning protein, the stop codon that was left between the biobricks had to be removed using site-directed mutagenesis. Finally, the new biobrick was cloned into Top10 competent E. coli cells and expression could be confirmed by observing the colonies. The plasmid was sent for sequencing to verify the plasmid sequence and to validate the part.
SELEX
SELEX or Systematic Evolution of Ligands by Exponential Enrichment is a screening technique based on selecting specific targets from a large pool of random oligonucleotides which could be a mixture of RNA, DNA, single or double-stranded, by an iterative process of separation and amplification. The products of SELEX are DNA or RNA aptamers that mimic the properties of antibodies. They can further bind to a big variety of molecules like proteins, peptides, drugs, organic molecules or even metal ions (Chai et al., 2011).
Figure 3: How SELEX binds to it's target
The reason why we used aptamers as our detection method this year is that aptamers are more flexible in their development and application. Aptamers are also easier to biochemically manipulate by means of adding functional groups, such as in our case through adding PCDA. The main principle of making aptamers remains the same despite having a different target: The random pool of DNA is incubated with the target. After the incubation, the binding complex is separated from the unbound sequences. In order to have the DNA sequences only, it then gets eluted. The last step is PCR amplification of the sequences, which then concludes a SELEX cycle. This process is repeated until the sequences are specific to the target. (Sefah et al., 2010).
Figure 4: Flowchart of SELEX
Cell-SELEX
The first part of the experiment was to create an aptamer that can detect C. acnes. C.acnes is a bacteria known for causing acne, but at the same time also belongs to the natural flora of the skin (Spittaels et al., 2020). For our experiment we used two types of C. acnes: CCUG 1794 T Cutibacterium acnes and CCUG 6369 Cutibacterium acnes were used for negative and positive control. At first, we incubated a random pool of DNA with our CCUG 6369 C. acnes cells after measuring the right amounts of cells. Next, we centrifuged the cell-aptamers mixture and removed the supernatant. As a result, we now only had the cells-aptamers complex. We then eluted the aptamers using heat shock and centrifugation at 14.000 rpm.
Next, we amplified the aptamers with PCR. The end product was a double-stranded DNA with the antisense sequence having a biotinylated primer. This primer was used so we can separate the sense from the antisense sequence using streptavidin beads and NaOH elution. At the end of the first round, we had only a pool of aptamers. In the second round, we incubated the pool of aptamers from the previous round,with the positive and negative bacteria and then repeated the steps from the first round. The protocol we used was an optimized version of the protocol from Sefah et al. (2010) and Simaeys et al. (2010).
Figure 5: How the bacteria finds and binds to the surface targets
LTA-SELEX
The second step of our experiment was to create an aptamer that is specific for Lipoteichoic acid (LTA), which is a molecule that exists in the membrane of all gram-positive bacteria (Poxton, 2015). In order to do that, we purchased LTA from S. aureus and using the carbodiimide method, we bound it to beads containing active carboxyl groups. We used the beads as a means of mobilization of the LTA and to make separation of the target-aptamer complex during the SELEX procedure easier. In order to obtain our LTA aptamer, we used the same method as explained above, but we replaced the cells with our LTA-beads as a positive control. For the negative control, we used the unbound beads.
Figure 6: Gram positive bacteria finding and binding to lipoteichoic acid
Protein-A Aptamer Testing
The last step of our experiment was to test our aptamer for protein A, a protein that can be found in the membrane of S. aureus. While an aptamer for the specific protein already exists (Stoltenburg et al., 2016), we wanted to make sure that it binds on our specific target before testing it. In order to do that we used HPLC with a protein A column and protein A-bound beads. Unfortunately, none of these methods yielded any results, which is why we tried another approach: We coated wells with protein A and incubated them with protein A aptamers attached to a fluorophore. Eventually, we detected the fluorescence, indicating that our aptamer is not capable of binding protein A.
References
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Hermanson, G. T. (2013). Zero-Length Crosslinkers. Bioconjugate Techniques, 259–273. https://doi.org/10.1016/B978-0-12-382239-0.00004-2
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