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

Optogenetic Control of Gene Expression in Corynebacterium glutamicum01:28

Optogenetic Control of Gene Expression in Corynebacterium glutamicum

136
Take a culture of Corynebacterium glutamicum, transformed with a plasmid carrying a fluorescent reporter gene downstream of the ribonucleic acid anti-terminator or RAT sequence, in a multi-well plate.During transcription, the RAT-RNA forms a stem-loop structure that blocks RNA polymerase, halting reporter gene expression.The plasmid also constitutively expresses LicV, a fusion protein comprising the RAT-RNA-binding protein, LicT, and the blue-light-sensitive VVD domain.In darkness, LicV remains...
136
A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction04:18

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction

1.1K
Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.
1.1K
Molecular Cloning09:55

Molecular Cloning

389.8K
Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called...
389.8K
Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes09:51

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

9.7K
The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug...
9.7K
Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 513:17

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

15.1K
A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.
15.1K
Reproductive Cloning01:27

Reproductive Cloning

32.6K
Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT).
Somatic Cell Nuclear Transfer
In SCNT, an egg cell is taken from an animal and its nucleus is removed, creating an enucleated egg. Then a somatic...
32.6K

You might also read

Related Articles

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

Sort by
Same author

Effect of Empagliflozin as an Add-On Therapy on Decongestion and Renal Function in Patients With Diabetes Hospitalized for Acute Decompensated Heart Failure: A Prospective Randomized Controlled Study.

Circulation. Heart failure·2021
Same author

Two cases of gastric adenocarcinoma with enteroblastic differentiation resected by endoscopic submucosal dissection.

Clinical journal of gastroenterology·2021
Same author

Effects of anti-thrombotic drugs on all-cause mortality after upper gastrointestinal bleeding in Japan: A multicenter study with 2205 cases.

Digestive endoscopy : official journal of the Japan Gastroenterological Endoscopy Society·2021
Same author

Efficacy of Sodium Carboxymethylcellulose Compared to Sodium Hyaluronate as Submucosal Injectant for Gastric Endoscopic Submucosal Dissection: A Randomized Controlled Trial.

Digestion·2021
Same author

Prognostic value of impaired hepato-renal function and liver fibrosis in patients admitted for acute heart failure.

ESC heart failure·2021
Same author

Incremental prognostic value of cardiac metaiodobenzylguanidine imaging over the co-morbid burden in acute decompensated heart failure.

ESC heart failure·2021

Related Experiment Video

Updated: Jan 19, 2026

Optogenetic Control of Gene Expression in Corynebacterium glutamicum
01:28

Optogenetic Control of Gene Expression in Corynebacterium glutamicum

Published on: October 29, 2025

136

Temperature-sensitive cloning vector for Corynebacterium glutamicum.

Jun Nakamura1, Sohei Kanno, Eiichiro Kimura

  • 1Fermentation and Biotechnology Laboratories, Ajinomoto CO., Inc., Kawasaki-shi, Kanagawa 210-8681, Japan. jun_nakamura@ajinomoto.com

Plasmid
|July 11, 2006
PubMed
Summary

Researchers developed a temperature-sensitive plasmid for Corynebacterium glutamicum. This tool enables conditional gene disruption in this industrially vital bacterium by controlling plasmid replication with temperature changes.

More Related Videos

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments
04:18

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Published on: May 17, 2024

1.1K
Molecular Cloning: Principle, Procedure, and Applications
09:55

Molecular Cloning: Principle, Procedure, and Applications

Published on: April 29, 2023

389.8K

Related Experiment Videos

Last Updated: Jan 19, 2026

Optogenetic Control of Gene Expression in Corynebacterium glutamicum
01:28

Optogenetic Control of Gene Expression in Corynebacterium glutamicum

Published on: October 29, 2025

136
Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments
04:18

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Published on: May 17, 2024

1.1K
Molecular Cloning: Principle, Procedure, and Applications
09:55

Molecular Cloning: Principle, Procedure, and Applications

Published on: April 29, 2023

389.8K

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Corynebacterium glutamicum is an industrially significant bacterium.
  • Efficient genetic tools are crucial for its manipulation and study.
  • Existing plasmids may lack conditional replication for precise genetic control.

Purpose of the Study:

  • To construct a temperature-sensitive replication system for the pBL1 cryptic plasmid in Corynebacterium glutamicum.
  • To develop a novel cloning vector for conditional gene disruption in C. glutamicum.
  • To identify the specific mutations responsible for temperature sensitivity.

Main Methods:

  • In vitro mutagenesis of the pSFK6 shuttle vector using hydroxylamine.
  • Transformation of Corynebacterium glutamicum cells with mutagenized plasmids.
  • Screening for plasmids with temperature-dependent replication stability.
  • DNA sequencing and site-directed mutagenesis to pinpoint causative mutations.

Main Results:

  • Isolation of a temperature-sensitive mutant plasmid, p48K, replicating at 25°C but not at 34°C.
  • Identification of four substitutions in the Rep protein coding region of p48K.
  • Demonstration that a single G-to-A transition (Pro-47 to Ser) in the Rep protein confers temperature sensitivity.
  • Pro-47 is a conserved residue in related plasmid Rep proteins.

Conclusions:

  • A functional temperature-sensitive replication system for C. glutamicum has been engineered.
  • The p48K plasmid provides a valuable tool for conditional gene disruption in C. glutamicum.
  • This vector facilitates research and genetic engineering in this important industrial microorganism.