Excitation Spectra for Wildtype GFP?

[Cathal Garvey]

Hey all,
I'm going to be working with a construct containing wildtype GFP, with
the original peptide sequence from A.victoria, and I'm hoping someone
here would know whether it's feasible to excite with Blue light (which I
think you can do with normal GFP?) or if I'll have to use UV?

Thanks
Cathal

[Josiah Zayner]

That should work. 

Any reason you used wtGFP and not one of the engineered versions?

[Cathal Garvey]

Patents. >:(
Thanks. According to a ref on wikipedia, folding is impaired in the WT
protein at 37C, so hopefully growing at 30C will help prevent that.
Photostability is bad too, so will have to be sparing in my illumination
of the samples..

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[Sebastian S. Cocioba]

Would it be feasible to reverse engineer an open egfp gene? Some point mutations would keep the sequence from being identical, no?

Sebastian S Cocioba
CEO & Founder
New York Botanics, LLC

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[Patrik D'haeseleer]

It's not just a matter of a couple of point mutations to make the sequence non-identical. Gene patents can claim pretty much any level of sequence homology - and they frequently do, essentially leaving it up to court challenges to decide what level of homology is justified.

Patrik

[xmort]

Actually I find the excitation /emission properties of wt GFP easier to work with in home/garage settings. I use UV LEDs for excitation, they have emission in 390-405nm range, ie exactly where you need them.  You can even buy ready to use UV LED flashlights for less than 10 bucks. For imaging I use green or yellow photographic filter, sturdy tripod and long exposure. I also find using old manual-everything lenses well fit for the purpose, because they don't reset when you need to reset your camera for changing batteries/card etc. 

here is one example

http://www.flickr.com/photos/morinkovo_fotky/3757039367/in/photostream

The excitation and emmision peaks are sufficiently far from each other (395-509 nm) , that you can easily block all the excitation light from your  image with inexpensive set of filters. It is much more difficult with most of the engineered version with 488ex/509em peaks to filter off the blue light from your image. You might need a dichroic mirror or some really good filter, which can be expensive. 

As fir the patent issue - are you really sure that eGFP is protected for your purpose while the wild type is exempt? I am not familiar with this particular issue, but most of the time all sequences hybridising with patented sequence or with more than say 90% protein sequence identity are covered by the patent. 

good luck tomas

Dne čtvrtek, 22. listopadu 2012 15:26:39 UTC+1 Cathal napsal(a):

[Cathal Garvey]

Huh, I'd been exciting with blue and using an orange filter (literally just using the Pearl Biotech illuminator/filter rig for this): would that not work well for blue excitation, green emission? Will go look for some UV LEDs to test with, too.

AFAIK, the patent on wildtype GFP ran out some years back, but patents remain on newer variants, mostly owned by MIT if memory serves. Even if the patents on new ones specify a method such as hybridisation (although that's typical of older patents, I find; pre-sequencing), the expiry of a patent covering the exact sequence I'm working with would offer me hard protection in the form of obvious prior art (as if nature itself wasn't good enough prior art..).

I eagerly await the expiry of the patents on eGFP and other staples of modern biotech, but for now it's just yet another lovely example of patents crushing even rudimentary innovative use.

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[xmort]

As long as you don't want to commercialize your final construct/product of  that construct, I wouldn't bother much with patent protection of GFP. In most countries patent infringement in academic and  non-for profit research is either tolerated or even explicitly made  possible by the law.  Even if you do plan to comemrcialize it eventually, the eGFP variant might expire by then so its kind of OK to use it.  Most generic companies work on patented drugs years before the original patent expires, so that they can market the generic version the day patent actually expires. 

[Cathal Garvey (Android)]

Ah, but I'm explicitly planning to sell it open sourcey, so both me and my customers would be at risk..

xmort <mor...@ueb.cas.cz> wrote:

As long as you don't want to commercialize your final construct/product of  that construct, I wouldn't bother much with patent protection of GFP. In most countries patent infringement in academic and  non-for profit research is either tolerated or even explicitly made  possible by the law.  Even if you do plan to comemrcialize it eventually, the eGFP variant might expire by then so its kind of OK to use it.  Most generic companies work on patented drugs years before the original patent expires, so that they can market the generic version the day patent actually expires. 

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