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

Fates of Pyruvate01:20

Fates of Pyruvate

8.3K
Pyruvate is the end product of glycolysis, where glucose is oxidized to pyruvate, simultaneously reducing NAD+ to NADH. Two molecules of ATP are also produced by substrate-level phosphorylation.
In aerobic organisms, pyruvate is metabolized via the citric acid cycle to produce reduced coenzymes NADH and FADH2. These coenzymes are then oxidized in the electron transport chain to produce ATP and, in the process, regenerate the NAD+ and FAD. As seen in some cell types and organisms, fermentation...
8.3K
Glycolysis: Pay-off Phase01:25

Glycolysis: Pay-off Phase

9.7K
So far, glycolysis has cost the cell two ATP molecules and produced two small, three-carbon sugar molecules. These molecules will proceed through the second half of the pathway, and sufficient energy will be extracted to pay back the two ATP molecules used as an initial investment and produce a profit for the cell of two additional ATP molecules and two even higher-energy NADH molecules.
Step 1 - 5: Glycolysis Preparatory Phase
The first phase of glycolysis has 5 steps where the glucose is...
9.7K
Glycolysis01:23

Glycolysis

Glycolysis, the Embden-Meyerhof pathway, is a central metabolic pathway involved in glucose catabolism. It is highly conserved across most organisms, reflecting its fundamental role in cellular energy production. This process occurs in the cytoplasm and can function both in the presence and absence of oxygen, making it versatile for various organisms and environmental conditions.Stages of GlycolysisGlycolysis is a ten-step pathway that converts glucose into pyruvate, generating a net gain of...
Pyruvate Oxidation01:15

Pyruvate Oxidation

158.3K
After glycolysis, the charged pyruvate molecules enter the mitochondria via active transport and undergo three enzymatic reactions. These reactions ensure that pyruvate can enter the next metabolic pathway so that energy stored in the pyruvate molecules can be harnessed by the cells.
First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+...
158.3K
ATP Yield01:31

ATP Yield

68.6K
Cellular respiration produces 30 - 32 ATP per glucose molecule. Although most of the ATP results from oxidative phosphorylation and the electron transport chain (ETC), 4 ATP are gained beforehand (2 from glycolysis and 2 from the citric acid cycle).
The ETC is embedded in the inner mitochondrial membrane and is comprised of four main protein complexes and an ATP synthase. NADH and FADH2 pass electrons to these complexes, which pump protons into the intermembrane space. This distribution of...
68.6K
ATP Energy Storage and Release01:31

ATP Energy Storage and Release

9.1K
ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
One example of energy coupling using ATP involves a...
9.1K

You might also read

Related Articles

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

Sort by
Same author

Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis.

Journal of visualized experiments : JoVE·2025
Same author

Cell-Free Protein Synthesis as a Method to Rapidly Screen Machine Learning-Generated Protease Variants.

ACS synthetic biology·2025
Same author

Rewireable Building Blocks for Enzyme-Powered DNA Computing Networks.

Journal of the American Chemical Society·2024
Same author

New Chloramphenicol Derivatives with a Modified Dichloroacetyl Tail as Potential Antimicrobial Agents.

Antibiotics (Basel, Switzerland)·2021
Same author

Modeling and control of transverse coupled bunch mode levels in Indus-2 using artificial neural network.

The Review of scientific instruments·2021
Same author

Horizontal fracture of anterior mandible.

BMJ case reports·2020
Same journal

A Framework for the In Vivo Production of Extensively Engineered Thiopeptides.

ACS synthetic biology·2026
Same journal

A Highly Stringent Split Intein-Mediated DHFR Selectable Marker Enables Efficient Development of High-Producing CHO Cells for Therapeutic Proteins.

ACS synthetic biology·2026
Same journal

Breaking the Stability-Activity-Selectivity Trilemma in Unspecific Peroxygenase through Computation-Based Cross-Regional Combinatorial Mutagenesis.

ACS synthetic biology·2026
Same journal

Sequential Plasmid Curing and Genome Editing in <i>Escherichia coli</i> Nissle 1917.

ACS synthetic biology·2026
Same journal

An Explainable Deep Learning Framework Integrating DNA Sequence and Transcription Initiation Signals for Gene Expression Prediction.

ACS synthetic biology·2026
Same journal

A Multitask Prediction Framework for CircRNAs, Drugs, and Diseases Based on Multi-View Information Integration and Graph Contrastive Learning.

ACS synthetic biology·2026
See all related articles

Related Experiment Video

Updated: Jun 4, 2025

OnePot PURE Cell-Free System
08:25

OnePot PURE Cell-Free System

Published on: June 23, 2021

8.4K

ATP Regeneration from Pyruvate in the PURE System.

Surendra Yadav1, Alexander J P Perkins1, Sahan B W Liyanagedera1

  • 1Centre for Engineering Biology, Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, U.K.

ACS Synthetic Biology
|January 4, 2025
PubMed
Summary
This summary is machine-generated.

Researchers enhanced cell-free protein synthesis using the PURE system by adding a novel ATP regeneration pathway. This improved system boosts protein production and is compatible with commercial kits.

Keywords:
ATP regenerationPUREcell-free protein synthesissynthetic biologysynthetic cellssynthetic metabolism

More Related Videos

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
14:42

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Published on: September 23, 2021

4.7K
An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes
08:40

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Published on: November 21, 2016

29.6K

Related Experiment Videos

Last Updated: Jun 4, 2025

OnePot PURE Cell-Free System
08:25

OnePot PURE Cell-Free System

Published on: June 23, 2021

8.4K
Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
14:42

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Published on: September 23, 2021

4.7K
An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes
08:40

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Published on: November 21, 2016

29.6K

Area of Science:

  • Biochemistry
  • Synthetic Biology

Background:

  • The Protein synthesis Using Recombinant Elements (PURE) system enables cell-free protein synthesis.
  • Enhancing ATP regeneration is crucial for optimizing PURE system efficiency.

Purpose of the Study:

  • To integrate a novel ATP regeneration system into the PURE system.
  • To evaluate the impact of this new system on protein synthesis yield and reproducibility.

Main Methods:

  • Incorporated pyruvate oxidase, acetate kinase, and catalase for ATP regeneration from pyruvate and phosphate.
  • Tested the system's performance with varying phosphate concentrations.
  • Assessed protein yield using mCherry as a model protein.
  • Validated results across homemade and commercial PURE systems.

Main Results:

  • Successfully generated acetyl phosphate for *in situ* ATP rephosphorylation.
  • Achieved high protein yields (up to 233 μg/mL mCherry) with a combined ATP regeneration system, a 78% increase.
  • Demonstrated that high phosphate concentrations (∼10 mM) do not inhibit protein synthesis.
  • Showed reproducibility and compatibility with commercial PURExpress.

Conclusions:

  • A novel, efficient ATP regeneration pathway was successfully integrated into the PURE system.
  • This enhancement significantly boosts cell-free protein synthesis yields.
  • The approach is rational, reproducible, and applicable to synthetic biology and synthetic cell construction.