Cell-free Expression
Cell-free Expression - Related Videos
10:34
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
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Cited by 2 •
2010
This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.
01:58
Cell Specific Gene Expression
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2020
Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
02:43
Transfection of an Inducible Gene Expression System into Cells Expressing a Bacterial Effector Protein
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2025
Source: Berens, C. et al. Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling. J. Vis. Exp. (2015)This video demonstrates the use of an inducible expression system to identify where the bacterial effector protein CaeB disrupts the host apoptotic cascade. It outlines the steps for transfecting CaeB-expressing cells with a tetracycline-responsive system to induce pro-apoptotic gene expression in order to identify the point of...
09:03
HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins
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Cited by 16 •
2015
Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.
03:32
Cell-Free Protein Expression Using a Bacterial Crude Cell Extract
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2025
Source: Wiegand, D. J., et al. Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens. J. Vis. Exp. (2019).This video demonstrates the use of a bacterial cell-free protein expression system based on Vibrio natriegens crude lysate to perform coupled in vitro transcription and translation. It highlights the preparation of a master mix with essential components and the step-by-step synthesis of functional proteins from a DNA template.
FAQs
The reaction’s output depends on which parts of gene expression are supplied and which template is introduced. A DNA or RNA template directs the process, while ribosomes and translation enzymes support protein production. Nucleotides, amino acids, and an energy source sustain these molecular activities, allowing researchers to examine RNA output, protein output, or transcription followed by translation.
Cell lysates provide a broader mixture of biological machinery, whereas purified systems contain defined molecular components. This distinction affects how explicitly the reaction can be specified and controlled, although both formats can support expression. Researchers can therefore select a lysate-based or component-defined setup according to whether they need a complex source of machinery or more controlled experimental conditions.
Because no viable culture must be maintained, experiments can focus on the genetic construct and reaction conditions rather than on keeping cells alive. This makes the approach useful for rapid study and engineering of gene function. It also supports testing outside the constraints of intact-cell maintenance while retaining the transcription and translation machinery needed to evaluate expression.
A circuit can be introduced as a DNA or RNA construct and evaluated through the RNA or protein it generates in the reaction. Researchers can prototype designs, compare genetic constructs, and observe whether the intended expression output is produced. Because the reaction is controlled and does not require viable cultures, multiple design ideas can be examined rapidly.
A basic workflow begins by selecting a DNA or RNA template, combining it with a lysate or purified expression machinery, and supplying the required nucleotides, amino acids, enzymes, ribosomes, and energy source. The reaction then supports transcription, translation, or both, depending on the design. Researchers examine the resulting RNA or protein as the experimental output.
Its supported uses include protein production, genetic-circuit prototyping, biosensor development, enzyme engineering, and rapid evaluation of genetic constructs. The method is also relevant when researchers need to study gene function under defined conditions. These applications make it useful in biological and synthetic biology workflows focused on designing, testing, and evaluating genetic systems.