In article <mab-0605962321320001 at f180-086.net.wisc.edu>,
mab at plantpath.wisc.edu wrote:
> ...I am looking for the
> new version of GFP that is much brighter than the original.
There are a couple of GFP variants that you might be interested in. The
most obvious is the S65T mutant described by Heim et.al. (1995) Nature
373:663-4. I am using this construct and it makes E.coli quite green when
driven by a T7 promotor.
Recently Crameri et.al. (1996) Nature Biotechnology 14:315-318 described a
GFP variant which they report is 45x brighter as the wt GFP, or 6-8x
brighter than S65T. I wrote to request this mutant; I include the
response I got from Pim Stemmer at the end of this post.
Both of these GFP variants have the advantage that their excitiation peak
is around the 488nm emmission line of an Argon-Ion laser. Their emmission
is reported to be around 510nm. These spectral characteristics match
common filter sets used for FITC fairly well.
There is also a brighter blue GFP variant, Y66H/Y145F, described by Heim &
Tsien (1996) Curr Biol 6:178-182. Its absorbance peak is ~380nm, and it
emits around 445nm. You might want to consider this if you're trying
double-labelling.
Check out Gibco-BRL and Clontech catalogs. I know that these 2 companies
sell GFP related products, but I have no idea what their current offerings
are. Otherwise I'd contact the authors of the papers describing the
mutants you are interested in.
jamie
--
Jamie White
Light Microscopy Group
Cell Biophysics Programme
European Molecular Biology Laboratory
Meyerhofstrasse 1, Postfach 10.2209
D-69117 Heidelberg Germany
+49 6221 / 387 367 voice
+49 6221 / 387 242 fax
finger jwhite at phenix.embl-heidelberg.de
********************
Here's the response I got regarding the c3 variant of Crameri et. al.:
X-Sender: jwhite at popserver.embl-heidelberg.de
Mime-Version: 1.0
Date: Wed, 08 May 1996 10:13:14 +0200
To: jwhite
From: Pim Stemmer <Pim_Stemmer at qmgate.affymax.com>
Subject: RE>request for c3 GFP mutant
Dear colleague,
Thank you for your interest in our GFP mutant constructs that were developed
by gene shuffling technology.
Affymax Research Institute has formed an independent unit, called Maxygene, to
focus on commercial application of gene shuffling technology, including
research reagents such as GFP derivatives.
We are distributing the prokaryotic and the eukarytic GFP constructs for US$
200 each. They are supplied as freeze dried plasmids (5 ug each; pBAD-GFP
cycle 3 and Alpha+GFP cycle 3). Both are supplied with diskettes containing
the full sequence of both plasmids and GFP as textfiles. No special expression
host is required. Our results were published and the vectors described by
Crameri et al., Nature Biotechnology 14:315-319 (1996). The bacterial vector
we used was described by Guzman et al., J. Bacteriol. 177:4121-4130 (1995),
and the eukaryotic vector by Whitehorn et al., Bio/Technology 13:1215-1219 (
1995). Useful restriction sites are listed below.
Please let me know if you are interested in obtaining one or both vectors.
We will need the shipping and billing address, as well as a purchase order
number.
Upon receipt of your order we will send you the materials by overnight mail,
and mail the invoice separately to your institutionUs billing address.
Sincerely -
Willem P.C. Stemmer, Ph.D.
Andreas Crameri,
Maxygen / Affymax Research Institute
4001 Miranda Avenue
Palo Alto, CA 94304.
Ordering and technical information:
phone: 415-812-8818
fax: 415-424-0832
e-mail: pim_stemmer at qmgates.affymax.com
Restriction sites flanking GFP:
Alpha+GFP cycle3:
Xho2-XhoI-XbaI-NheI(--GFP gene--) EcoRI-EcoRV-HinDIII-ClaI-BamHI
pBAD-GFP cycle 3:
BamHI-NdeI-NheI(--GFP gene--)EcoRI-KpnI-XmaI-SmaI-BamHI-XbaI-SalI-PstI-SphI-
HindIII.
A full sequence restriction map is provided with each plasmid.
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