SELEX Protocols and SELEX Troubleshooting Guides
During our time in the lab, we worked diligently on optimising every step of our in-vitro SELEX and cell SELEX protocols.
Moreover, we curated extensive troubleshooting guides for both in-vitro SELEX and cell SELEX. These guides encompass profoundly detailed SELEX protocols, together with troubleshooting tips for the key steps and other related protocols. We also included a "Before the experiment" section, aiming to guide future iGEM teams on how to order the essentials needed for SELEX, with special focus on the library and primer ordering.
Check them out!
BioBrick
AMIL-GFP - LABELLED PROTEIN A
To that demonstrate the fuctionality of our rapid test, we based our proof of concept 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 on the target. When the binding is performed with immobilised targets, functional groups recognised by the aptamer might partake in the immobilisation, which ultimately impairs their rocognition ability (2).
Moreover, when designing binding assays, we sought to mimic the biosensor prototype, where the aptamers would be bound to the PCDA vesicles and 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.
Figure 1: Vector map of pSB1C3 containing the final version of the amilGFP-protein A BioBrick (BBa_K4071000).
Human Practices Handbook
The human practices handbook, originally created by iGEM Stockholm 2017, has been updated by this year's iGEM Stockholm team together with iGEM Chalmers-Gothenburg. The pandemic has pushed the teams to be creative in terms of collaborating, organising events and reaching out to the public. In times of change, we saw the opportunity to adapt the handbook to pass on our knowledge and experience to future teams.
A chapter about the Nordic iGEM conference, NiC, was added in the handbook by iGEM Chalmers-Gothenburg which was one of the organizers of this year's NiC. The chapter can also be useful for teams organising other conferences or large events.
See the updated version of the handbook here.
Finance Handbook
The first ever Finance Handbook was drafted by iGEM Stockholm 2021. The need for this document was realised pretty early during the project since knowledge transfer from previous teams proved to be a pain point. This handbook contains
- List of main tasks for finance members
- Important grants that the team must apply to and their application deadlines
- Previous company sponsorships and respective contact person details
- Email templates
- Other important learnings from year 2021
You can download and read the handbook below
References
Lakhin AV, Tarantul VZ, Gening L. Aptamers: problems, solutions and prospects. Acta Naturae. 2013 ; 5 (4 (19) ).
Pfeiffer F, Mayer G. Selection and biosensor application of aptamers for small molecules. Frontiers in chemistry. 2016 Jun 15 ; 4 : 25.