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

Indigoidine synthetases: Versatile biocatalysts in nature and the laboratory.

Current opinion in chemical biology·2026
Same author

Evolutionary insights and guidelines to achieve effective and high-yield non-ribosomal peptide and polyketide engineering.

Current opinion in microbiology·2026
Same author

Identification and Evaluation of dibasic piperidines as novel cell wall inhibitors against <i>Mycobacterium tuberculosis</i>.

bioRxiv : the preprint server for biology·2026
Same author

A rapid combinatorial assembly method for gene cluster characterisation illuminates glidobactin biosynthesis.

Synthetic and systems biotechnology·2025
Same author

Novel TdsD nitroreductase: characterization of kinetics and substrate specificity.

Biotechnology letters·2025
Same author

Optimized chemogenetic ablation and regeneration of enteric nervous system neurons in zebrafish.

bioRxiv : the preprint server for biology·2025

Related Experiment Video

Updated: Apr 23, 2026

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly
10:18

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Published on: February 9, 2015

37.2K

A gain-of-function positive-selection expression plasmid that enables high-efficiency cloning.

Gareth A Prosser1, Elsie M Williams, Jack A Sissons

  • 1School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.

Biotechnology Letters
|September 27, 2014
PubMed
Summary

Directed enzyme evolution requires efficient cloning. A new vector, pUCXKT, ensures 100% cloning efficiency for mutant gene libraries, maximizing efforts in biocatalysis research.

More Related Videos

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
08:31

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

13.9K
Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes
07:10

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes

Published on: September 29, 2023

4.7K

Related Experiment Videos

Last Updated: Apr 23, 2026

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly
10:18

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Published on: February 9, 2015

37.2K
Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
08:31

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

13.9K
Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes
07:10

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes

Published on: September 29, 2023

4.7K

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Directed enzyme evolution is crucial for enhancing biocatalytic properties.
  • Low-throughput screening necessitates high cloning efficiency in mutant gene libraries.
  • Screening empty plasmids wastes valuable research resources.

Purpose of the Study:

  • To develop a novel expression vector that maximizes cloning efficiency.
  • To facilitate the screening of improved enzyme variants from mutant gene libraries.
  • To overcome limitations of existing positive selection vectors.

Main Methods:

  • Development of pUCXKT, a gain-of-function positive selection expression vector.
  • Utilizing a specialized downstream PCR primer for gene amplification.
  • Restoring regulatory and genetic elements for co-expression of a kanamycin resistance marker.

Main Results:

  • pUCXKT demonstrated 100% cloning efficiency for inserted genes.
  • High-level expression of cloned genes was achieved.
  • The vector design is adaptable to various vector backbones and marker genes.

Conclusions:

  • pUCXKT significantly improves cloning efficiency in directed evolution workflows.
  • This vector enables more effective screening of beneficial enzyme variants.
  • The adaptable design offers broad utility in molecular cloning and protein engineering.