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Reporter Genes

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.

Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

PCR

PCR

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DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

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Applicability of Control Materials To Support Gene Promoter Characterization and Expression in Engineered Cells Using

Ana Fernandez-Gonzalez¹, Simon Cowen², Juhyun Kim³

¹Molecular and Cell Biology Team, National Measurement Laboratory, LGC, Teddington, Middlesex, TW11 0LY, United Kingdom.

Analytical Chemistry

|March 31, 2022

Summary

This summary is machine-generated.

This study developed standardized control materials and used digital PCR (dPCR) to precisely measure small gene expression changes. These tools aid in validating engineered biological systems and advancing standardization efforts in synthetic biology.

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Area of Science:

Synthetic Biology
Molecular Biology
Biotechnology

Background:

Standardization of components and validation methods is crucial for engineering biological systems.
Current efforts focus on developing reliable tools for measuring gene expression accurately.
Accurate quantification of small fold-changes is essential for validating engineered systems.

Purpose of the Study:

To develop a panel of control materials for validating engineered biological systems.
To validate digital PCR (dPCR) as a high-accuracy method for quantifying small gene expression changes.
To identify sources of error in measuring small ratios in biological systems.

Main Methods:

Development of a control material panel with varying copy number differences of reporter genes (GFP and RFP) as DNA or RNA.
Quantification of gene and transcript copy numbers using digital PCR (dPCR).
Validation of dPCR in a bacterial gene circuit model to assess gene expression changes.

Main Results:

dPCR accurately quantified gene and transcript number changes down to 1.05-fold differences.
dPCR precisely identified small changes in gene expression in response to promoter stimulation in a bacterial model.
Identified key sources of error contributing to measurement uncertainty in biological systems.

Conclusions:

The developed control materials and validated dPCR method enhance the engineering biology toolkit.
This work supports ongoing standardization efforts in synthetic biology by providing reliable measurement tools.
Improved accuracy in measuring small gene expression changes facilitates robust validation of engineered biological systems.