Workflow
June
- First PCDA synthesis using different concentrations for proof of concept. We tried to make a stock solution which did not work due to low solubility.
- Preparation of LB and Glycerol stocks
July
- Inoculation of C. acnes
- Buffer preparations
- LTA-bead conjugation
- Library preparation (resuspension)
- Cloning was started with the aim of practicing and creating a biobrick with protein A with a GFP attached. A plasmid from the 2019 distribution kit was used but it did not work, although the same plasmid from the 2021 distribution kit worked.
- 1st round of SELEX
- No results, extensive troubleshooting and reruns.
- Increased amount of PCR cycles, different template concentrations
- Noticed we had bought the wrong reverse primer, it had biotin on the wrong terminal.
- Another mistake that we noticed was that our protein A was stored at RT instead of the - 35 to - 25 ℃ that is recommended on the package. According to instructors, this was nothing to be concerned about.
- A new PCDA solution was created with a concentration of 1 mM - the same concentration as is required for coupling. This solution became deep blue after polymerisation. This was subsequently used for all conjugation attempts.
- Aptamer coupling with PCDA was tested with different concentrations of PCDA in the coupling process, from a volume of 50 µL PCDA as suggested in the protocol to a maximum volume of 237.5 µL. A higher PCDA concentration seemed to provide a higher yield after dialysis, meaning that more PCDA vesicles were conjugated to the same amount of aptamers.
- Protein A coating of plates for protein A aptamer testing. The aptamer coupled to PCDA was tested in the protA wells for binding and colour change, which did not yield any colour change. Further investigation was needed.
- PCDA testing with heat. Heat was used to test if a colour change is possible: the PCDA solution did indeed turn to a bright orange when subjected to a burner. The PCDA was also tested with UV light, which did induce a colour change, although very slowly. The solution turned to a deep purple when left under UV light overnight.
August
- Start of cell SELEX using C. acnes as targets.
- Protein A aptamer testing using affinity chromatography. A Mabselect column used for IgG purification was used to test the binding of our protein A aptamer. The aptamer solution ran through the column when the sample was applied, meaning that no binding took place. An attempt was made using magnetic protein A beads, with the same results. It is possible that the aptamer binds to a different part of protein A than is accessible in the column and on the beads.
- Finally, since none of the SELEX runs worked, and after extensive troubleshooting using different annealing temperatures, working concentrations and cycle numbers, we had a meeting with our advisor Dimitri. We concluded that the libraries required HPLC purification which we did not choose when ordering. Thus, a new library was ordered with HPLC purification.
September
October
Lab Notes
1st of October is our deadline for lab work.
Protocols
Selex
DVS Aptamer Selection Protocol
LTA Aptamer Selection Protocol
PCDA
Culture
The above protocol is an open access iGEM protocol by Paul Rutten from The University of Oxford et al.
Cloning
Monarch® DNA Gel Extraction Kit Protocol
QIAquick® PCR Purification Kit
The above protocol is repeated in the Gel Extraction protocol, this is simply the official company card.
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Birds aren't real.
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Pranav Ballaney, 2020
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In text citation for another research article with a DOI. Allen & Sheridan, 2015
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