BIOBRICK
AMIL-GFP - LABELLED PROTEIN A
To that demonstrate the fuctionality of our rapid test, we based our proof of concept (link) on testing the PCDA detection method with a published aptamer. To this aim, we conjugated the PCDA vesicles with an aptamer that targets protein A (S. aureus surface protein), and attempted to test its sensitivity and binding affinity.
Ideally, aptamer binding assays are performed with the target in solution. Since aptamers are small in size (20-60 nucleotides) (1), they are able to fit into clefts and gaps within the target’s surface. Consequently, each aptamer recognises a very specific but also limited region in the target. When the binding is performed with immobilised targets, it might occur that the functional groups or structures recognised by the Aptamer are the ones involved in the immobilisation. (2) Therefore, the recognition ability might be impaired.
Moreover, we seek to mimic the rapid test prototype (link?), where the aptamers would be bound to the PCDA vesicles and would recognise the bacteria in solution.
Unfortunately, the BioBrick registry lacks plasmids containing protein A labelled with GFP and/or a chromoprotein that we could purify and use in our experiments.
For this reason, we decided to create a BioBrick containing Protein A labelled with a reporter protein. The idea was to create a recombinant protein A visible with the naked eye. Thus, we chose to clone protein A together with the yellow chromoprotein Amil-GFP to create a new BioBrick.
Hopefully, our amil-GFP - labelled protein A will be useful for future iGEM teams working with S.aureus-related projects.
insert picture of final BioBrick - we don’t have it yet REFERENCES: sorry the first one doesn’t have doi for whatever reason 1- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890987/ 2- https://doi.org/10.3389/fchem.2016.00025
Figure 1: Our Biobrick
SELEX protocols
During our time in the lab we worked very hard on optimising every step of our in-vitro SELEX and cell SELEX protocols (links).
Here you can find detailed in-vitro SELEX and cell SELEX protocols together with troubleshooting tips for every step.
Troubleshooting
Still need to re-write protocols and recapitulate the tips from everyone in the wet lab I think I should create a different document for each protocol so it can be downloaded as a PDF —> more useful
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.
In text citation for a website with an author. Author, n.d.