Overview
The goal of this year's iGEM team was to create a detection method based on aptamers bound to PCDA vesicles. When the aptamers bind to its target due to mechanical stress the PCDA will change color indicating that the target is present. Quantification of the intensity of the color 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). Due to their unique chromatic properties, they have been used as a detection method. 10,12-Pentacosadiynoic acid (PCDA) belongs in the family of polydiacetylenes and in its primary form yields a blue color but under the exposure of heat (thermochromism), mechanical stress (mechanochromism) or solvent (solvatochromism) can change color from blue to red (Lebègue et al., 2018). For our experiments mechanochromism was of great importance, because as mentioned before binding of the aptamer causes mechanical stress, resulting in color change.
Formation of PCDA vesicles
In order to create the PCDA vesicles we dissolved the PCDA in chloroform and then we evaporated the chloroform using gentle N2 flow. Then, we dissolved the PCDA in water using sonication. The end product was an emulsion with a white color. The next step was exposure in UV light to induce polymerization resulting in color 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 4oC until further use.
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) which is used in order to stabilize the reactive intermediate.
The reason we chose this method is because the PCDA has carboxy groups and the aptamer is a small DNA sequence which has amine groups. The protocol we followed was based on a paper by Wu et al., (2012).
Bacteria Cultivation
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 and they can bind to a big variety of molecules like proteins, peptides, drugs, organic molecules or even metal ions (Chai et al., 2011).
The reason why we used aptamers as our detection method this year is because aptamers are more flexible in their development and application compared to antibodies and are easier biochemically manipulated in means of adding functional groups like in our case with adding PCDA. The main principal 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 and then eluted in order to have the DNA sequences only. The last step is PCR amplification of the sequences and that concludes a SELEX cycle. This process is repeated until the sequences are specific for the target. (Sefah et al., 2010).
Cell-SELEX
The first part of the experiment was to create an aptamer that can detect C. acnes a bacteria known for causing acne but at the same time belongs in the natural flora of the skin (Spittaels et al., 2020). In order to do that we used two types of C. acnes, more specifically, CCUG 1794 T Cutibacterium acnes and CCUG 6369 Cutibacterium acnes were used for negative and positive control respectively. At first, we incubated a random pool of DNA with our CCUG 6369 C. acnes cells after measuring the right amounts of cell. Next, we centrifuged the cell-aptamers mixture and removed the supernatant and as a result we had only the cells-aptamers complex. Then we 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 used the pool of aptamers from the previous round, we incubated it with the positive bacteria and with the negative and then we followed the same steps as the first round. The protocol we used was optimized from the protocol from Sefah et al. (2010) and (Simaeys et al., 2010).
LTA-SELEX
The second step of our experiments 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 carboxy groups. We used the beads as a means of mobilization of the LTA and because it was easier to separate the target-aptamer complex during the SELEX procedure. In order to obtain our LTA aptamer, we used the same method as before, but we replaced the cells with our LTA-beads as a positive control and for negative control we used the unbound beads.
Protein-A Aptamer Testing
The last step of our experiments was to test our protein A aptamer. Protein A is a protein that exists in the membrane of S. aureus and an aptamer for the specific protein already exists (Stoltenburg et al., 2016). However before testing it we wanted to make sure that it binds on our target. 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 so we tried another approach. We coated wells with protein A and we incubated it with protein A aptamer attached to a fluorophore. Finally we detected the fluorescence, establishing that our aptamer is 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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