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Related Experiment Videos

Avoiding and controlling double transformation artifacts.

Moshe Goldsmith1, Csaba Kiss, Andrew R M Bradbury

  • 1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.

Protein Engineering, Design & Selection : PEDS
|June 19, 2007
PubMed
Summary
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Standard control experiments can authenticate new protein variants by ruling out artifacts from double transformants, which are common and can skew results. Implementing these protocols ensures the reliability of protein variant selection and characterization.

Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Biochemistry

Background:

  • Library selection and site-directed mutagenesis are common methods for protein variant isolation.
  • Artifacts from 'double transformants' (cells with multiple plasmids) can compromise experimental results.
  • The prevalence and impact of double transformants are often underestimated in protein variant studies.

Purpose of the Study:

  • To describe standard control experiments for authenticating novel protein variants.
  • To specifically address and mitigate artifacts arising from double transformants.
  • To provide a reliable protocol for validating variants obtained from heterogeneous gene repertoires.

Main Methods:

  • Utilizing standard molecular cloning and plasmid isolation techniques.

Related Experiment Videos

  • Implementing subcloning procedures to verify plasmid integrity.
  • Performing functional assays to confirm the activity or binding properties of isolated variants.

    • Main Results:

    • Demonstrated that double transformants are more common than previously recognized.
    • Established that standard controls effectively rule out artifacts from double transformants.
    • Validated a protocol for ensuring the authenticity of selected protein variants.

    Conclusions:

    • Standardized control experiments are essential for the accurate authentication of protein variants.
    • The described protocols effectively prevent artifacts caused by double transformants.
    • This methodology is crucial for reliable protein engineering and functional studies, especially for variants selected for enzymatic activity or binding.