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Related Experiment Videos

Protein evolution by codon-based random deletions.

Joel Osuna1, Jorge Yáñez, Xavier Soberón

  • 1Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología/UNAM, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.

Nucleic Acids Research
|October 2, 2004
PubMed
Summary

A new method, codon-based random deletion (COBARDE), enables targeted deletion of gene codons. This molecular tool facilitates protein engineering by exploring novel sequence spaces and evolving protein functions.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Synthetic Biology

Background:

  • Gene manipulation is crucial for understanding protein function and evolution.
  • Existing methods for gene modification may lack efficiency or combinatorial capacity.
  • Targeted deletion of codons offers a precise approach to alter protein structure and function.

Purpose of the Study:

  • To develop a novel method for in-phase codon deletion within specific gene regions.
  • To create a robust, automatable, and tunable system for combinatorial mutagenesis.
  • To demonstrate the utility of this method in evolving protein function.

Main Methods:

  • Development of the codon-based random deletion (COBARDE) method.
  • Utilizing oligonucleotide assembly and transient orthogonal protecting groups in chemical synthesis.

Related Experiment Videos

  • Applying the COBARDE method to TEM-1 beta-lactamase to alter substrate specificity.

    • Main Results:

    • The COBARDE method successfully deleted complete codons in a random and combinatorial manner.
    • Engineered TEM-1 beta-lactamase variants exhibited altered substrate specificity, including ceftazidime resistance.
    • Functional variants with deleted residues in the critical omega-loop region were identified.

    Conclusions:

    • The COBARDE method provides a powerful new tool for molecular engineering.
    • This technique expands the accessible sequence space for protein evolution studies.
    • COBARDE facilitates the discovery of novel protein functions through targeted genetic modifications.