Fluorescent tags are widely used for microscopy and expression studies – but it wasn’t so long ago that this everyday tool was unheard of. In this article we’ll talk about how GFP came to be, and what it means for you.
Green fluoresecent protein, or GFP, was first identified in a fluorescent jellyfish, Aequorea victoria. Osamu Shimomura purified GFP and described the biophysics of how it fluoresces. A few years later, Martin Chalfie reported the expression of this protein in E. coli and C. elegans. Roger Tsien is responsible for designing variants on the protein – single amino acid changes that yielded cyan, blue and yellow fluorescent proteins, and the enchanced green protein (EGFP) that is commonly used today. Shimomura, Chalfie and Tsien were awarded the Nobel Prize for Chemistry in 2008 for their work in the discovery and development of this tool. What made GFP such a game changer is the fact that it’s “auto-catalytic” – it doesn’t need any co-factors or enzyme processing to fluoresce – so it can be easily used in a wide variety of organisms.
The major applications for GFP proteins are microscopy based, since its primary value is as a visual marker for protein detection. Here are a few of the most popular ways to use GFP:
1. Translational fusion
One of the most common uses is a fusion marker, where the GFP open reading frame is cloned downstream of your favorite ORF, so that it is translated as one long protein, fusing your favorite protein to GFP. That way, wherever your protein is expressed you will see green fluoresence. This can be used in still images and is striking in images of live cells, as you can track the location and movement of proteins.
2. Transcriptional fusion
GFP can also be used in “transcriptional fusion”, where the expression of a gene and GFP are driven off the same promoter, but with an intervening stop codon. In this case, cells expressing the first gene will fill with soluble GFP – resulting in easy detection of the particular cells expressing your protein.
3. FLIP and FRAP
FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) rely on the fact that a single GFP molecule emits fluorescent light when it’s excited, but cannot do so indefinitely. Eventually it either bleaches out or stops emitting. So, to study the dynamics of a GFP-labeled protein, you can bleach a small area of a cell and determine how long it takes fluorescently labeled protein to “leak” back into the bleached area (FRAP), or how much fluorescence decreases overall in the rest of the cell as the bleached proteins diffuse (FLIP).
4. FRET
FRET (fluorescence resonance energy transfer) is based on the different excitation and emission spectra of the different variations on GFP. In this case, two proteins are labeled with two different fluorophores, which are carefully selected so the emission spectrum of the first overlaps the excitation spectrum of the second. The cells are then imaged using a laser that excites only the first fluorophore – so the second only lights up if the two proteins are in close enough proximity that the first fluorophore sets off the second.
Check out some of the seminal papers written about GFP:
Green fluorescent protein as a marker for gene expression.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC.
Science. 1994 Feb 11;263(5148):802-5.
Wavelength mutations and posttranslational autoxidation of green fluorescent protein.
Heim R, Prasher DC, Tsien RY.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501-4.
A post from bitesizebio.
Thursday, May 19, 2011
Wednesday, May 11, 2011
Antibiotic selection is always a tool of great importance in molecular biology. But Ampicillin as an antibiotic, everyone faces a problem to some extent. The following artical is from BitesizeBio gives some helpful tips to overcome this problem.
Ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it serves it’s purpose, there can be problems using this selection marker if the user is unaware of it’s limitations. This article provides a quick overview of what these limitations are and how to avoid them.
The basis of ampicillin selection is the hydrolysis and inactivation of the antibiotic by beta-lactamase expressed from the plasmid-borne bla gene. Here’s the problem: beta-lactamase is secreted by the bacteria. The resulting build-up of extracellular beta-lactamase can inactivate the ampicillin in the culture medium, removing the selective pressure, if the culture is not handled properly.
In liquid cultures, this means that a portion (possibly a very large portion) of the cells no longer have the plasmid, giving poor yielding plasmid preps, protein expression etc. On agar plates, ampicillin degradation can lead to the formation of satellite colonies on transformation plates. Satellite colonies are very small colonies of cells that have not taken up the plasmid that form around a large colony that has taken up the bla-containing plasmid. The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony. The satellites are not necessarily a problem as they will not grow when transferred to a medium containing fresh ampicillin.
So how do you avoid plasmid loss when using ampicillin as a selection marker? Here’s how:
## Don’t allow liquid cultures to saturate for too long. I would recommend never growing cultures higher than OD600=3 (in LB)
## If you are using a starter culture, always pellet and re-suspend the starter culture in fresh, antibiotic-free medium before innoculating the main culture. This is to remove the secreted beta-lactamase from the medium.
## Use a higher ampicillin concentration if you are experiencing problems. I would recommend using 200 micrograms per mL or higher. This makes it harder for the beta-lactamase to inactivate all of the ampicillin and is especially useful for avoiding satellite formation.
## For the same reason, never use old ampicillin stocks or plates as the ampicillin will have broken down somewhat, giving a reduced effective ampicillin concentration.
If all else fails, switch to carbenicillin selection. This antibiotic is also inactivated by beta-lactamase but more slowly than ampicillin is. For this reason carbenicillin selection is far more effective than ampicillin. Unfortunately it is much more expensive!
Ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it serves it’s purpose, there can be problems using this selection marker if the user is unaware of it’s limitations. This article provides a quick overview of what these limitations are and how to avoid them.
The basis of ampicillin selection is the hydrolysis and inactivation of the antibiotic by beta-lactamase expressed from the plasmid-borne bla gene. Here’s the problem: beta-lactamase is secreted by the bacteria. The resulting build-up of extracellular beta-lactamase can inactivate the ampicillin in the culture medium, removing the selective pressure, if the culture is not handled properly.
In liquid cultures, this means that a portion (possibly a very large portion) of the cells no longer have the plasmid, giving poor yielding plasmid preps, protein expression etc. On agar plates, ampicillin degradation can lead to the formation of satellite colonies on transformation plates. Satellite colonies are very small colonies of cells that have not taken up the plasmid that form around a large colony that has taken up the bla-containing plasmid. The satellites form because the beta-lactamase released by the bla-expressing colony degrades the ampicillin in the vicinity of the colony. The satellites are not necessarily a problem as they will not grow when transferred to a medium containing fresh ampicillin.
So how do you avoid plasmid loss when using ampicillin as a selection marker? Here’s how:
## Don’t allow liquid cultures to saturate for too long. I would recommend never growing cultures higher than OD600=3 (in LB)
## If you are using a starter culture, always pellet and re-suspend the starter culture in fresh, antibiotic-free medium before innoculating the main culture. This is to remove the secreted beta-lactamase from the medium.
## Use a higher ampicillin concentration if you are experiencing problems. I would recommend using 200 micrograms per mL or higher. This makes it harder for the beta-lactamase to inactivate all of the ampicillin and is especially useful for avoiding satellite formation.
## For the same reason, never use old ampicillin stocks or plates as the ampicillin will have broken down somewhat, giving a reduced effective ampicillin concentration.
If all else fails, switch to carbenicillin selection. This antibiotic is also inactivated by beta-lactamase but more slowly than ampicillin is. For this reason carbenicillin selection is far more effective than ampicillin. Unfortunately it is much more expensive!
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