Difference between revisions of "Part:BBa K4159003:Design"
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We used a DNA synthesising machine from CODEX DNA to synthesise 32 od our DARPins with this BBa_K4159003 spacer. The synthesising went well and there was no problem of tandem repeats or similar with the spacer in the sequence.  
 
We used a DNA synthesising machine from CODEX DNA to synthesise 32 od our DARPins with this BBa_K4159003 spacer. The synthesising went well and there was no problem of tandem repeats or similar with the spacer in the sequence.  

Revision as of 16:50, 1 October 2022


Spacer sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

No changes were needed to the sequence. This sequence was taken from the pBx1-VHH_template3-3XMyc-Spacer [1], and placed at the end of the fragment for ribosome display of the DARPin construct. This spacer is derived from the pBx1-VHH_template3-3XMyc-Spacer plasmid from Chen et al. (2021). The spacer has been used in ribosome display for the selection of nanobodies. As our project is related to DARPin (design ankyrin repeat protein) selection, which is in the same size range (~14kDa) as the nanobodies, we were confident that the spacer was good for our selection. The spacer encodes for 72 amino acid residues, which should be long enough to “push out” the DARPin peptide structure from the ribosome.

Check out our part BBa_K4159000 for a detailed description of how we used it in our composite part.

Characterization: We performed in total three ribosome display experiments. Two of them were affinity selecting our 42 DARPins against the biotinylated AIP1 target, while one of them was affinity selecting our GFP-targeting DARPin against GFP. For the ribosome display containing the GFP-targeting DARPin we added additionally a small aliquot of DARPin #12. This would confirm that the GFP-targeting DARPin would bind the GFP target in higher quantities.


alt text

We used a DNA synthesising machine from CODEX DNA to synthesise 32 od our DARPins with this BBa_K4159003 spacer. The synthesising went well and there was no problem of tandem repeats or similar with the spacer in the sequence.

After performing the ribosome display selection, we sent the cDNA for sequencing and got the results of some DARPins attached (for more information about sequencing and the results, visit our results page https://2022.igem.wiki/aalto-helsinki/results ), which shows that the spacer was sufficient for this ribosome display experiment and is aligned with the data also from the literature.

Source

[1] Chen X, Gentili M, Hacohen N, Regev A. A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies. Nat Commun. 2021 Sep 17;12(1):5506. doi: 10.1038/s41467-021-25777-z. PMID: 34535642; PMCID: PMC8448731.

References