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

Spore inoculum size impacts substrate degradation and sporulation but not the secretome during colonization of whole yellow pea (Pisum sativum) by Aspergillus oryzae.

International journal of food microbiology·2026
Same author

Reverse prediction of carbohydrate esterase polysaccharide targets.

Biotechnology for biofuels and bioproducts·2026
Same author

A Hadza-enriched <i>Prevotella/Segatella</i> xyloglucanase shows sequence conservation and functional specialization.

Gut microbes reports·2026
Same author

3D-Bioprinted Marine Bacteria for the Degradation of Polyhydroxybutyrate Bioplastics.

ACS applied polymer materials·2026
Same author

Bridging Simulation and Sustainability: Laccase Immobilization on Bio-Polymeric Hybrids for Degradation of 17α-Ethinylestradiol in Water Systems.

ACS omega·2026
Same author

3D Printed In Vitro Engineered Living Material Models for Antimicrobial Development.

ACS applied bio materials·2026

Related Experiment Video

Updated: Apr 7, 2026

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
14:06

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Published on: November 12, 2012

47.2K

Essential validation methods for E. coli strains created by chromosome engineering.

Sriram Tiruvadi Krishnan1, M Charl Moolman1, Theo van Laar1

  • 1Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft, 2628 CJ The Netherlands.

Journal of Biological Engineering
|July 4, 2015
PubMed
Summary

Accurate validation of engineered E. coli strains is crucial. This study presents essential methods like Southern blotting and phage contamination assays to ensure genomic integrity and reliable strain construction.

Keywords:
Cell shape analysisChromosome engineeringEBU plate assayEscherichia coliGrowth curve analysisP1 phage transductionRecombineeringStrain validation

More Related Videos

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing
09:44

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Published on: December 23, 2022

3.1K
Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes
08:13

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Published on: September 1, 2018

18.2K

Related Experiment Videos

Last Updated: Apr 7, 2026

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
14:06

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Published on: November 12, 2012

47.2K
Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing
09:44

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Published on: December 23, 2022

3.1K
Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes
08:13

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Published on: September 1, 2018

18.2K

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Chromosome engineering uses homologous recombination to modify microbial genomes, particularly in Escherichia coli.
  • Techniques like λ-Red recombination (recombineering) and P1 phage transduction are key for introducing multiple genomic insertions.
  • Reliable validation is essential due to potential errors during strain construction.

Purpose of the Study:

  • To outline comprehensive validation methods for chromosomally engineered E. coli.
  • To address limitations of standard verification techniques like PCR and DNA sequencing.
  • To ensure accuracy and reliability in genetic modification studies.

Main Methods:

  • Utilizing Polymerase Chain Reaction (PCR) and DNA sequencing for initial verification.
  • Employing Southern blotting to detect extraneous DNA insertions.
  • Implementing cross-streak agar and Evans Blue-Uranine (EBU) plate assays for temperate phage contamination detection.
  • Assessing cell growth and morphology for additional validation.

Main Results:

  • Standard PCR and sequencing may not detect all chromosomal engineering errors.
  • Southern blotting effectively identifies non-specific insertions during recombineering.
  • EBU plate assays provide a novel method for detecting temperate phage contamination in E. coli.
  • Growth and shape analysis serves as a complementary validation step.

Conclusions:

  • A suite of validation techniques ensures reliable verification of engineered E. coli strains.
  • Additional assays beyond PCR and sequencing are necessary for comprehensive error detection.
  • Methods discussed are broadly applicable to various chromosome engineering strategies in E. coli.