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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
In vitro Mutagenesis01:16

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Standardizing cassette-based deep mutagenesis by Golden Gate assembly.

Nicolas Daffern1, Irene M Francino-Urdaniz1, Zachary T Baumer1

  • 1Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA.

Biotechnology and Bioengineering
|September 26, 2023
PubMed
Summary

We developed a standardized Golden Gate assembly method for creating large, user-defined protein-encoding libraries. This efficient protocol generates millions of variants, advancing protein engineering capabilities.

Keywords:
Golden Gatecassettesdeep mutagenesislibrariesoligo poolsprotein engineering

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Protein Engineering

Background:

  • Existing methods for constructing large, mutagenized protein-encoding libraries using Golden Gate assembly are often system-specific and lack standardization.
  • This limits the broad applicability and scalability of generating diverse protein variants for research and development.

Purpose of the Study:

  • To present a standardized Golden Gate assembly method for building user-defined, deeply mutagenized protein-encoding libraries.
  • To demonstrate the scalability and efficiency of this method for generating a high number of unique protein variants.

Main Methods:

  • A standardized Golden Gate assembly protocol was developed using a 25 μL reaction volume with 40 fmol of input DNA.
  • The method accommodates double-stranded DNA (dsDNA) cassettes generated from degenerate oligonucleotides or oligonucleotide pools.
  • Scalability of reaction volume and input DNA concentration was assessed without loss in transformation efficiency.

Main Results:

  • The standardized method successfully generated libraries on the order of 1 × 10^6 members from a single reaction.
  • Scaling up reaction volume and input DNA concentration did not compromise transformation efficiency.
  • Custom, user-defined libraries ranging from 10^4 to 10^7 unique protein-encoding variants were constructed for two orthogonal protein engineering systems.

Conclusions:

  • The presented standardized Golden Gate method provides a robust and scalable platform for constructing diverse protein-encoding libraries.
  • This approach simplifies and enhances the process of protein engineering by enabling the generation of large, user-defined variant libraries.
  • The availability of a detailed protocol and general-use destination vectors facilitates broader adoption in scientific research.