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

Plasmids01:28

Plasmids

1.2K
Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
1.2K
Chromosome Structure02:40

Chromosome Structure

25.9K
A functional eukaryotic chromosome must contain three elements: a centromere, telomeres, and numerous origins of replication.
The centromere is a DNA sequence that links sister chromatids. This is also where kinetochores, protein complexes to which spindle microtubules attach, are constructed after the chromosome is replicated. The kinetochores allow the spindle microtubules to move the chromosomes within the cell during cell division.
Telomeres consist of non-coding repetitive nucleotide...
25.9K
DNA Bacteriophages01:26

DNA Bacteriophages

819
Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...
819
Replication in Prokaryotes01:32

Replication in Prokaryotes

27.6K
DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
27.6K
Replication in Prokaryotes02:35

Replication in Prokaryotes

96.9K
Overview
96.9K

You might also read

Related Articles

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

Sort by
Same author

Development and optimization of a glycerol-based fed-batch strategy for the production of peroxidases with Komagataella phaffii under the P<sub>DF</sub> promoter.

Microbial cell factories·2026
Same author

Comprehensive assessment of fungal peroxygenases for the selective synthesis of 25-hydroxyvitamins D<sub>2</sub> and D<sub>3</sub>.

Biotechnology for biofuels and bioproducts·2026
Same author

MAPS: a marker-free genome integration system for Komagataella phaffii enabling co-production of natural pigments and industrial enzymes.

Metabolic engineering·2026
Same author

Enhanced secretion of thermostable phytases from Myceliophthora thermophila by Komagataella phaffii.

Microbial cell factories·2026
Same author

Enhanced genetic stability and expression control in growth-decoupled continuous two-stage E. coli fermentations using plasmid-dependent thyA auxotrophic selection.

Microbial cell factories·2025
Same author

Next-generation stress-inducible Komagataella phaffii promoter variants.

Microbial cell factories·2025

Related Experiment Video

Updated: Jan 16, 2026

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
09:46

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Published on: February 25, 2010

47.1K

Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.

Carsten Pichler1, Florian Weiss1, Anton Glieder2

  • 1Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.

Methods in Molecular Biology (Clifton, N.J.)
|October 1, 2025
PubMed
Summary

Stable episomal plasmids are crucial for microbial biotechnology. This study demonstrates the successful use of ARS1-containing plasmids in Komagataella phaffii, enabling antibiotic-free selection and diverse applications like CRISPR/Cas gene expression.

Keywords:
ARS plasmidAutonomously replicating sequenceEpisomal expressionKomagataella phaffiiPARS1Pichia pastoris

More Related Videos

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
05:30

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Published on: January 23, 2019

14.4K
Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
15:28

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

Published on: November 16, 2012

14.9K

Related Experiment Videos

Last Updated: Jan 16, 2026

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
09:46

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Published on: February 25, 2010

47.1K
Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
05:30

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Published on: January 23, 2019

14.4K
Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
15:28

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

Published on: November 16, 2012

14.9K

Area of Science:

  • Microbial biotechnology
  • Molecular biology
  • Yeast genetics

Background:

  • Plasmids are essential tools for recombinant DNA delivery in microbial biotechnology for producing valuable compounds.
  • Instability of circular episomal plasmids in Komagataella phaffii necessitates genomic integration, limiting transformation rates and expression uniformity.
  • Existing episomal plasmid systems in P. pastoris (formerly K. phaffii) lack the stability and high transformation rates seen in Saccharomyces cerevisiae.

Purpose of the Study:

  • To develop and validate a stable episomal plasmid system for Komagataella phaffii.
  • To overcome limitations of genomic integration methods for gene expression and strain engineering.
  • To enable high-throughput applications and reduce reliance on selective markers.

Main Methods:

  • Utilized ARS1 autonomously replicating sequences (ARS) for plasmid replication and partitioning in K. phaffii.
  • Implemented various selection markers including antibiotic resistance, auxotrophy, and carbon source utilization.
  • Applied ARS1 plasmids for antibiotic-free selection, knockout strain complementation, and transient CRISPR/Cas gene expression.

Main Results:

  • Demonstrated successful and stable episomal maintenance of ARS1-containing plasmids in K. phaffii.
  • Achieved high transformation rates and low clonal variability comparable to S. cerevisiae episomal systems.
  • Validated the utility of ARS1 plasmids for diverse applications, including antibiotic-free selection and CRISPR/Cas systems.

Conclusions:

  • ARS1-containing plasmids offer a stable and efficient alternative to genomic integration in K. phaffii.
  • This system enhances K. phaffii's utility in research and industrial applications by improving transformation efficiency and expression consistency.
  • The developed plasmid system broadens the toolkit for genetic manipulation and protein production in K. phaffii.