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

PCDA Synthesis

PCDA Aptamer Conjugation

PCDA Concentration

Culture

Ampicillin Solutions

CFU Determination

Calibration Protocol

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

Cloning and Transforming

Gel Extraction

QIAquick® PCR Purification Kit

QIAquick® Gel Extraction Kit

The above protocol is repeated in the Gel Extraction protocol, this is simply the official company card.

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Pranav Ballaney, 2020

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Citations

In text citation for a research article with a DOI. Rosano et al., 2019

In text citation for another research article with a DOI. Allen & Sheridan, 2015

In text citation for a book with no DOI. Ingalls, 2013

In text citation for a website with institutional author. TNAU Agritech Portal, n.d.

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References

1. Allen, M. J., & Sheridan, S. C. (2015).

   Mortality risks during extreme temperature events (ETEs) using a distributed lag non-linear model.

   International Journal of Biometeorology 62(1), 57-67.

   CrossRefGoogle ScholarBack to text

2. Rosano, A., Bella, A., Gesualdo, F., Acampora, A., Pezzotti, P., Marchetti, S., ... & Rizzo, C. (2019).

   Investigating the impact of influenza on excess mortality in all ages in Italy during recent seasons (2013/14-2016/17 seasons).

   International Journal of Infectious Diseases 88, 127-134.

   CrossRefGoogle ScholarBack to text

3. Ingalls, B. P. (2013).

   Mathematical modeling in systems biology: An introduction.

   MIT Press.

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4. Agriculture: Crop production: Sugarcane. TNAU Agritech Portal.

   (March 15, 2019). Retrieved on June 22, 2020. from https://google.com

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5. Author Name. (n.d.).

   Agriculture: Crop production: Sugarcane. TNAU Agritech Portal.

   Retrieved on June 22, 2020. from https://google.com

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