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Method Article
Florian Hollfelder
Department of Biochemistry, University of Cambridge
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1367-6312
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.
Keywords
directed evolution, protein engineering, mutagenesis, insertions and deletions, in vitro transposition
Figure 1
Figure 2
This is a list of supplementary files associated with this preprint. Click to download.
- pIDTet.gb
Supplementary file 1: full sequence and map of pID-Tet in GenBank format.
- pIDT7.gb
Supplementary file 2: full sequence and map of pID-T7 in GenBank format.
- pUC57TransDel.gb
Supplementary file 3: full sequence and map of pUC57-TransDel in GenBank format.
- pUC57TransIns.gb
Supplementary file 4: full sequence and map of pUC57-TransIns in GenBank format.
- pUC57Del2.gb
Supplementary file 5: full sequence and map of pUC57-Del2 in GenBank format.
- pUC57Del3.gb
Supplementary file 6: full sequence and map of pUC57-Del3 in GenBank format.
- pUC57Ins1.gb
Supplementary file 7: full sequence and map of pUC57-Ins1 in GenBank format.
- pUC57Ins2.gb
Supplementary file 8: full sequence and map of pUC57-Ins2 in GenBank format.
- pUC57Ins3.gb
Supplementary file 9: full sequence and map of pUC57-Ins3 in GenBank format.
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Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Jun, 2021
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Associated Publications
Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis
by Stephane Emond, Maya Petek, Emily J. Kay, +4
Nature Communications (10 July, 2020)
In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region
by Kalliopi Skamaki, Stephane Emond, Matthieu Chodorge, +7
Proceedings of the National Academy of Sciences (16 October, 2020)
10.1038/s41467-021-23450-z
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Florian Hollfelder
Department of Biochemistry, University of Cambridge
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1367-6312
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Jun, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.
Figure 1
Figure 2
This is a list of supplementary files associated with this preprint. Click to download.
- pIDTet.gb
Supplementary file 1: full sequence and map of pID-Tet in GenBank format.
- pIDT7.gb
Supplementary file 2: full sequence and map of pID-T7 in GenBank format.
- pUC57TransDel.gb
Supplementary file 3: full sequence and map of pUC57-TransDel in GenBank format.
- pUC57TransIns.gb
Supplementary file 4: full sequence and map of pUC57-TransIns in GenBank format.
- pUC57Del2.gb
Supplementary file 5: full sequence and map of pUC57-Del2 in GenBank format.
- pUC57Del3.gb
Supplementary file 6: full sequence and map of pUC57-Del3 in GenBank format.
- pUC57Ins1.gb
Supplementary file 7: full sequence and map of pUC57-Ins1 in GenBank format.
- pUC57Ins2.gb
Supplementary file 8: full sequence and map of pUC57-Ins2 in GenBank format.
- pUC57Ins3.gb
Supplementary file 9: full sequence and map of pUC57-Ins3 in GenBank format.
Loading...
Associated Publications
Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis
by Stephane Emond, Maya Petek, Emily J. Kay, +4
Nature Communications (10 July, 2020)
In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region
by Kalliopi Skamaki, Stephane Emond, Matthieu Chodorge, +7
Proceedings of the National Academy of Sciences (16 October, 2020)
10.1038/s41467-021-23450-z