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 Concept Videos

You might also read

Related Articles

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

Sort by
Same author

Carbon assimilation and regulation by the phosphotransferase system in Actinobacillus succinogenes.

Journal of applied microbiology·2025
Same author

Insights into lactose catabolism and its regulation in Actinobacillus succinogenes.

Journal of applied microbiology·2025
Same author

A Comparative Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Conventional Methods for the Diagnosis of Dermatophytes.

Cureus·2025
Same author

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

Cell metabolism·2024
Same author

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility.

PLoS computational biology·2022
Same author

Secondary Alcohol Dehydrogenases from Thermoanaerobacter pseudoethanolicus and Thermoanaerobacter brockii as Robust Catalysts.

Chembiochem : a European journal of chemical biology·2021

Related Experiment Video

Updated: May 2, 2026

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
11:45

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Published on: May 29, 2016

11.8K

Development of a markerless knockout method for Actinobacillus succinogenes.

Rajasi V Joshi1, Bryan D Schindler, Nikolas R McPherson

  • 1Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.

Applied and Environmental Microbiology
|March 11, 2014
PubMed
Summary

Researchers developed a markerless gene knockout method for Actinobacillus succinogenes, enhancing its succinate production capabilities. This new technique allows for efficient genetic engineering of this key industrial microorganism.

More Related Videos

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility
12:29

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Published on: March 11, 2022

2.1K
Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System
05:06

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

Published on: January 5, 2024

1.3K

Related Experiment Videos

Last Updated: May 2, 2026

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
11:45

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Published on: May 29, 2016

11.8K
Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility
12:29

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Published on: March 11, 2022

2.1K
Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System
05:06

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

Published on: January 5, 2024

1.3K

Area of Science:

  • Microbiology
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Actinobacillus succinogenes is a key organism for succinate production.
  • Improving succinate yield and productivity requires genetic engineering.
  • Existing genetic tools for A. succinogenes are limited.

Purpose of the Study:

  • To develop a novel markerless gene knockout method for Actinobacillus succinogenes.
  • To enable efficient genetic manipulation for enhanced succinate production.
  • To establish a reusable selection system for microbial engineering.

Main Methods:

  • Developed a markerless knockout system using natural transformation and electroporation.
  • Employed an Escherichia coli isocitrate dehydrogenase gene as a positive selection marker, leveraging A. succinogenes's glutamate auxotrophy.
  • Utilized Saccharomyces cerevisiae flippase recombinase (Flp) for marker removal and reuse.
  • Demonstrated efficient recombination with minimal homologous DNA (200 bp) and selection cassette flanking (1 kb).

Main Results:

  • Successfully created single and double knockout mutants in A. succinogenes.
  • Achieved efficient gene deletions, including fumarate reductase and pyruvate formate lyase.
  • Demonstrated the ability to introduce at least two consecutive deletions in the same strain.
  • Validated electroporation as an alternative transformation method.
  • Established direct selection of knockout mutants on agar plates using an enriched defined medium.

Conclusions:

  • The developed markerless knockout method is efficient and versatile for A. succinogenes genetic engineering.
  • This method significantly advances the potential for metabolic engineering of A. succinogenes for improved succinate production.
  • The reusable selection system and optimized recombination parameters facilitate rapid strain development.