Difference between revisions of "Protein coding sequences/Reporters"

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Introduction to Reporters

What are reporters? Why call them reporters and not fluorescent proteins, beta-galactosidase/Xgal assays or pigments? The answer is that in synthetic biology, they are often used as a mechanism to display information. If you design a biosensor system that is sensitive to substrate A, you ideally want a way of displaying these results without needing an additional assay. Using a reporter protein is a great way of achieving this property.

Reporter protein coding sequences encode proteins whose presence in the cell or organism is readily observed. For example, fluorescent proteins cause a cell to fluoresce when excited with light of a particular wavelength, luciferases cause a cell to catalyze a reaction that produces light, and enzymes such as beta-galactosidase convert a substrate to a colored product. Reporters are frequently used to quantify the strength or activity of upstream gene expression parts such as promoters and ribosome binding sites. Reporters, when fused in frame to other protein coding sequences, can also be used to identify where a protein is located in a cell or organism.

There are several different ways to measure or quantify a reporter depending on the particular reporter and what kind of characterization data is desired. Generally speaking, microscopy is useful for obtaining both spatial and temporal information on reporter activity, particularly at the single cell level. Flow cytometers are best suited for measuring the distribution in reporter activity across a large population of cells. Plate readers are generally best for taking population average measurements of many different samples over time.

Some iGEM teams have excellent projects that create new reporters, or make great use of existing ones. Please see the tables below for fluorescent proteins, chromoproteins, luciferase proteins and enzymes that produce colored substrates in the Registry.

Fluorescent proteins

GFP visualized under UV light.

Fluorescent proteins are convenient ways to visualize or quantify the output of a device or part. Many different fluorescent proteins are available from the Registry. The first fluorescent protein to be cloned and the most commonly used fluorescent protein is green fluorescent protein (GFP). GFP was cloned from jellyfish. Based on GFP, several investigators generated mutations that altered the spectral properties of GFP resulting in yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP).

RFP visualized in white light.

Red fluorescent protein (RFP), shown on the left, was isolated from coral. Note that the colonies appear red to the naked eye as well as fluoresce red. RFP is unrelated to the GFP family of fluorescent proteins. Several derivatives of RFP have been generated including mOrange and mCherry.

Fluorescence can be readily quantified using a microscope, plate reader or flow cytometer equipped to excite the fluorescent protein with the appropriate wavelength of light. Since since several different fluorescent proteins are available, multiple gene expression measurements can be made in parallel.

Luciferases

Luminescence can be readily quantified using a plate reader or luminescence counter. Luciferases are especially well-suited for measuring low levels of gene expression because cells tend to have little to no background luminescence in the absence of a luciferase.

Enzymes that produce colored substrates

Enzymes that produce colored substrates can be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers. Like luciferases, enzymes like β-galactosidase are good for measuring low levels of gene expression because they tend to amplify low signals.

Miscellaneous

References

<biblio>

1. Pardy pmid=7866866

</biblio>

Fluorescent proteins

<biblio>

1. Chalfie pmid=8303295
2. Prasher pmid=1347277
3. Cormack pmid=8707053
4. Tsien pmid=9759496
5. Andersen pmid=9603842
6. Leveau pmid=11698362
7. Iafolla pmid=18350571
8. Dong pmid=18352052
9. Bagh pmid=18352063
10. Baird pmid=11050229
11. Patterson pmid=15583657

</biblio>

Luciferase

<biblio>

1. Bronstein pmid=8080073

</biblio>

β-galactosidase

<biblio>

1. Griffith pmid=11779182
2. Villarejo pmid=4552692
3. Zamenhof pmid=4552986
4. Ullman pmid=1345751

</biblio> [http://openwetware.org/index.php?title=Beta-Galactosidase_Assay_%28A_better_Miller%29 Miller Assay on OpenWetWare] by Sean Moore