Part Collection

Our project has been about designing, testing, and expressing DARPins specific to our project. We have been working with two different DARPins, one that is a well-known DARPin that binds to GFP, and a DARPin that would bind to the signalling molecule AIP. Our collection is made up of the parts needed to test and express these DARPins. More information about the background of the projects can be found here. The whole collection has been also divided into two sub-collection to make it easier to understand the concept.

GFP DARPin collection

The first sub-collection is the GFP DARPin collection which has all parts needed for expressing and testing the DARPin in the binding assay.

The main part of this sub-collection is the GFP DARPin sequence. It has been derived from literature, and more complex characterization information can be found on the Main parts page (Hansen et al., 2017). The other major independent part of both sub-collections is of course the spacer sequence needed for the ribosome display. This spacer sequence is derived also from literature and has been used quite recently in experiments regarding the selection of nanobodies (Chen et al., 2021). The GFP DARPin sequence has then been put together in different composite parts, depending on the downstream process. The GFP DARPin for ribosome display construct consists then of the spacer, which is needed for successful selection and the appropriate promoter and ribosome binding site. The other composite part, which is the GFP DARPin for expression, does not then include the spacer, but has added tags for more efficient purification and also a terminator. This collection works as a good base for working with the GFP DARPin, and can also act as a good reference for other ribosome display constructs.

AIP-DARPin collection

The other sub-collection is then about the AIP-binding DARPin. The difference from the GFP binding DARPin collection is in the exact design of the DARPin. In this sub-collection, we haven't directly copied from the literature the DARPin sequence, so the success of binding is not certain. The modifications done in the DARPin sequence here are based on different papers (Schilling et al, 2014; Hansen et al. 2017; Seger et al., 2013) and give hopefully the expected result. The backbone of the AIP-binding DARPin is based still on a common consensus sequence, and only a few mutations have been done. More about the exact design and background can be found on the design page [link]. Similarly to the first sub-collection, this sub-collection is also made up of the actual AIP-binding DARPin that has been found to be the most successful, and the spacer sequence needed after the DARPin sequence.

For this sub-collection, we have also included our bioreporter sequences, as they have been necessary for the exact detection of the effect our DARPin has on the cellular system. To make our project work easier and faster, we decided to order the reporter parts as big as possible, to avoid unnecessary cloning work. For future teams to use these parts, they can be either ordered easily separately from our individual parts or as a composite part, as we did. The first reporter part is the part containing two promoters for the reporter to work. For this part, we have also done an extensive engineering cycle. The second part has the target genes for the reporter to work. The part that is not included in our part collection is the reporter protein gene, which has been GFP in our case. The part has not been included here, due to the fact that we used a plasmid backbone (pET28a-sfGFP), which already contained the GFP sequence. This gives also the opportunity for other teams to attach whichever reporter protein gene they want and does not necessarily have to be the GFP.

Conclusion

As a whole, the collection is quite diverse and can serve both as the control for future ribosome display assays or as the background knowledge for how to design all parts needed for this type of assays. All protocols and experiments done with these parts can be found in the protocols and experiments section.

References

  1. Chen X, Gentili M, Hacohen N, Regev A (2021)
    A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies
    Nature Communication 2021 Sep 17;12(1):5506
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  2. Schilling, J., Schöppe, J., & Plückthun, A. (2014)
    From DARPins to LoopDARPins: novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display
    Journal of molecular biology, 426(3), 691-721
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  3. Hansen S, Stüber JC, Ernst P, Koch A, Bojar D, Batyuk A, Plückthun A (2017)
    Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity
    Scientific Reports 2017 Nov 24;7(1):16292/i>
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  4. Seeger, M. A., Zbinden, R., Flütsch, A., Gutte, P. G., Engeler, S., Roschitzki-Voser, H., & Grütter, M. G. (2013)
    Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity
    Protein science, 22(9), 1239-1257.
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