Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Heat-inducible autolytic vector for high-throughput screening.

Lihua Xu1, Shuang Li, Chuan Ren

  • 1Tsinghua University, Beijing, China.

Biotechniques
|September 23, 2006
PubMed
Summary

Researchers developed heat-inducible autolytic vectors to efficiently lyse bacterial cells for enzyme screening. This method simplifies enzyme variant analysis in directed evolution, improving high-throughput screening processes.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Aperiodic activity modulation during visual oddball paradigm in clinical high-risk individuals and relationship with psychosis conversion.

Schizophrenia research·2026
Same author

Clarifying the "component-structure-function" mechanism in processed mozzarella cheese: the mechanistic role of fat, protein, and water interactions on quality and texture.

Food chemistry·2026
Same author

Advances in oncolytic virus delivery strategies and challenges in clinical translation.

Materials today. Bio·2026
Same author

Valence-specific oculomotor abnormalities induced by dynamic video stimuli as a robust biomarker for schizophrenia.

Asian journal of psychiatry·2026
Same author

Eye Movement Patterns as Robust Biomarkers for Schizophrenia Identification Using a Novel Data Transformation Approach.

Journal of eye movement research·2026
Same author

Dental pulp stem cell exosomes promote angiogenesis via the PI3K/Akt signaling pathway to treat androgenetic alopecia.

Stem cell research & therapy·2026

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • High-throughput screening in directed evolution requires efficient enzyme variant sampling.
  • Intracellular enzyme expression in hosts like Escherichia coli necessitates cell membrane disruption, which is often laborious and costly.
  • Current methods for cell lysis can be time-consuming and expensive, hindering rapid screening.

Purpose of the Study:

  • To develop a novel heat-inducible autolytic vector system to simplify bacterial cell lysis.
  • To enable efficient screening of enzyme variants in directed evolution.
  • To facilitate rapid extracellular assays of heterologous enzymes.

Main Methods:

  • Construction of heat-inducible autolytic vectors using the SRRz lysis gene cassette from bacteriophage lambda.

Related Experiment Videos

  • Utilized lambda cI857/pR promoter and its mutant c1857/pR(M) for inducible gene expression.
  • Inserted autolytic units into the pUC18 backbone and tested lysis efficiency using beta-galactosidase assays.
  • Incorporated Bacillus subtilis 168 lipase A gene to demonstrate extracellular enzyme assay utility.

    • Main Results:

    • The wild-type cI857/pR promoter achieved 96.3% lysis efficiency upon temperature shift from 28°C to 38°C.
    • The mutant cI857/pR(M) promoter allowed bacterial growth at 35°C and lysis at 42°C, but showed inconsistent lysis efficiency.
    • Expression of Bacillus subtilis lipase A using the autolytic vector resulted in 93.7% activity in the culture medium after heat induction.

    Conclusions:

    • The constructed heat-inducible autolytic vectors offer a robust and efficient method for bacterial cell lysis.
    • This system significantly streamlines the process of enzyme variant screening in directed evolution.
    • The vector's utility was confirmed for rapid extracellular assay of heterologous enzymes, enhancing biotechnological applications.