by Leslie Kent
Fun Rating: 5/5
Difficulty Rating: 2/5
What is the general purpose? During gene expression, DNA is transcribed to RNA, which is then translated to proteins. After translation, these proteins can do their jobs inside and even outside the cell. Cells can fine-tune gene expression in response to their environment. Scientists use assays that measure gene expression to study expression patterns, which help us better understand what different genes do.
Why do we use it? Fluorescent transcriptional reporters are commonly used by scientists to measure gene expression because they are relatively inexpensive in materials and require less time than other methods. They also generate quantitative data and allow measurement of the amount of protein produced instead of only the amount of RNA.
How does it work? Fluorescent transcriptional reporters use fluorescence to measure or report on transcription and gene expression. First, transcriptional reporters are formed by combining a piece of regulatory DNA, such as a promoter, and a reporter gene. Promoters are regions of DNA that tell RNA polymerase (a protein that transcribes DNA to RNA) where to bind (for example, right before a gene). For fluorescent transcriptional reporters, the reporter gene encodes a fluorescent protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP).
Image 1. A transcriptional reporter that is made up of a promoter and a reporter gene. Created by Leslie Kent with BioRender.com.
Next, the fluorescent transcriptional reporter is put into the cells of interest. As the cells express their own genes, they can also express the reporter gene, resulting in the production of fluorescent protein. Then, the amount of that fluorescent protein can be quantified by measuring the brightness of the fluorescent cells. When some cells are brighter than others, they have more fluorescent reporter protein, which means more promoter activity. This increase in activity tells us that the genes that are normally controlled by that promoter likely also have more gene expression.
Fluorescent transcriptional reporters have many applications. For example, if we feed some growing bacteria a different type of sugar, such as lactose instead of glucose, we might expect a change in gene expression. Specifically, we could hypothesize that the bacteria will respond by increasing expression of genes that encode proteins necessary for eating that specific kind of sugar. To test this hypothesis, we could make a fluorescent transcriptional reporter with the promoter that normally controls expression of genes necessary for eating lactose. Then, we can grow our bacteria with and without lactose and compare the fluorescence in these two conditions.
Image 2. The same promoter for the gene encoding the protein for eating lactose is used to promote transcription of the gene encoding the reporter protein. Created by Leslie Kent with BioRender.com.
If our hypothesis is correct, we would expect more fluorescence in the lactose condition, because the bacteria are transcribing genes for proteins that enable them to eat the lactose.
Image 3. A bacterial cell grown with lactose has more reporter GFP than a bacterial cell grown without lactose. More GFP results in more brightness, which we can measure with a plate reader. Created by Leslie Kent with BioRender.com.
As seen from our thought experiment, fluorescent transcriptional reporters are a useful tool for geneticists to monitor and quantify changes in gene expression, further advancing our understanding of genetics.
