From owner-fluorescent@net.bio.net Sat Jan 04 22:00:00 1997 Path: biosci!SCRIPPS.EDU!stevek From: stevek@SCRIPPS.EDU ("Steve A. Kay") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Postdoctoral Position in Molecular Neuroscience Date: 5 Jan 1997 10:52:55 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 22 Sender: daemon@net.bio.net Distribution: world Message-ID: <32CFF787.4A97@scripps.edu> NNTP-Posting-Host: net.bio.net Postdoctoral Position Molecular and Cellular Neuroscience Position available to study activity-dependent regulation of neuronal structure. Projects involve the use of digital imaging technology, neuronal cell culture, molecular biology, and protein biochemistry to examine signal transduction events that target cytoskeletal proteins of dendrites and synapses. Our laboratory=92s emphasis is on protein-protein interactions in living neurons. The laboratory is equipped for state-of-the-art confocal and conventional fluorescence microscopy and three-dimensional image processing. Candidates with experience in these and other techniques of cellular and molecular neurobiology may apply by sending a letter of interest, CV, and names of references to: Dr. Shelley Halpain Department of Cell Biology The Scripps Research Institute 10550 North Torrey Pines Road La Jolla, CA 92037 email: shelley@scripps.edu Scripps Home Page: www.scripps.edu From owner-fluorescent@net.bio.net Sun Jan 05 22:00:00 1997 Path: biosci!POST.QUEENSU.CA!lapointr From: lapointr@POST.QUEENSU.CA (Lapointe, Renee) Newsgroups: bionet.molbio.proteins.fluorescent Subject: (none) Date: 6 Jan 1997 16:39:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701070040.TAA28796@post.queensu.ca> NNTP-Posting-Host: net.bio.net info faq From owner-fluorescent@net.bio.net Sun Jan 05 22:00:00 1997 Path: biosci!agate!cgl!espinoza From: espinoza@cgl.ucsf.edu (Hernan Espinoza) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Counting GFP colonies Date: 6 Jan 97 22:24:52 GMT Organization: UCSF Computer Graphics Lab Lines: 13 Message-ID: References: <32D14DE4.6D81@umich.edu> NNTP-Posting-Host: socrates.ucsf.edu pboucher@UMICH.EDU (Paul Boucher) writes: >I am interested in counting colonies that stably express GFP for >cytotoxicity assessment studies. I want to be able to count colonies >that lack and express GFP. Is there an easier way of doing this than >counting the colonies under a fluorescent microscope? Perhaps some sort >of fluorescent dish scanner. I would appreciate any input. Thank-you. Do you need to count colonies, or would counting cells work? If so, you could use a FACS machine to count up cells expressing and not expressing GFP. It could give you better numbers and the you would only have to handle a few tubes instead of many plates. Well, that's my lame suggestion. -Hernan From owner-fluorescent@net.bio.net Sun Jan 05 22:00:00 1997 Path: biosci!UMICH.EDU!pboucher From: pboucher@UMICH.EDU (Paul Boucher) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Counting GFP colonies Date: 6 Jan 1997 11:09:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <32D14DE4.6D81@umich.edu> NNTP-Posting-Host: net.bio.net I am interested in counting colonies that stably express GFP for cytotoxicity assessment studies. I want to be able to count colonies that lack and express GFP. Is there an easier way of doing this than counting the colonies under a fluorescent microscope? Perhaps some sort of fluorescent dish scanner. I would appreciate any input. Thank-you. Laura Zerbe Rubsam From owner-fluorescent@net.bio.net Mon Jan 06 22:00:00 1997 Path: biosci!rutgers!news.sgi.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.tufts.edu!opal.tufts.edu!jstrassw From: jstrassw@opal.tufts.edu Newsgroups: bionet.molbio.proteins.fluorescent Subject: Globs of GFP fusion in cytosol? Date: 7 Jan 97 09:48:12 -0500 Organization: Tufts University - Medford, MA Lines: 13 Distribution: world Message-ID: <1997Jan7.094812@opal.tufts.edu> NNTP-Posting-Host: opal.tufts.edu Hello! I made a fusion of pEGFP (clontech) to my cDNA of unknown function. the resulting pattern is that of 10 or so bright globs in the cytoplasm of HeLa and C33A cells transiently transfected. I am concerned that this may represent the formationm of inclusion bodies due to overexpresession of an insoluble form of the fusion. On the other hand, it could represnt targeting to a specific structure within the cytoplasm. Thanks for your help, John Tufts U. Boston, USA From owner-fluorescent@net.bio.net Mon Jan 06 22:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!nih.gov!usenet From: bernard@elsie.nci.nih.gov (Bernard Murray) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Counting GFP colonies Date: 7 Jan 1997 18:44:33 GMT Organization: National Institutes of Health, Bethesda, MD Lines: 21 Distribution: world Message-ID: <5au5ih$633@light.nih.gov> References: <32D14DE4.6D81@umich.edu> NNTP-Posting-Host: ermintrude.nci.nih.gov X-Newsreader: WinVN 0.99.7 In article <32D14DE4.6D81@umich.edu>, pboucher@UMICH.EDU says... > >I am interested in counting colonies that stably express GFP for >cytotoxicity assessment studies. I want to be able to count colonies >that lack and express GFP. Is there an easier way of doing this than >counting the colonies under a fluorescent microscope? Perhaps some sort >of fluorescent dish scanner. I would appreciate any input. Thank-you. > Laura Zerbe Rubsam Could you get away with lysing the plate of cells and then comparing the fluorescence (in a fluorimeter) with protein or DNA concentration? You could then relate this to the results of GFP-negative and GFP-fully- expressing cells (measured in separate determinations). This is clearly the quick'n'cheap way of doing it. For more precise results you could use a FACS if you have one available. Has anyone tried modifying a Coulter Counter...? Bernard Bernard Murray, Ph.D. bernard@elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA) From owner-fluorescent@net.bio.net Mon Jan 06 22:00:00 1997 Path: biosci!POST.QUEENSU.CA!lapointr From: lapointr@POST.QUEENSU.CA (Lapointe, Renee) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Quantification of GFP and Expression at 33C Date: 7 Jan 1997 12:56:50 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701072057.PAA00257@post.queensu.ca> NNTP-Posting-Host: net.bio.net Hi, I am interested in the use of GFP for quantification of baculovirus promoter expression in a transient assay. What method of quantification of GFP could I use, and would it be possible to detect and quantify two different GFP variants under different promoters in the same cells. Could anyone refer me to specific references on quantification assays. Another concern that I have is the efficiency of the GFP at 33C. Does anyone have experience on this? Thank you for your help Renee Lapointe Department of Microbiology and Immunology Queen's university From owner-fluorescent@net.bio.net Tue Jan 07 22:00:00 1997 Path: biosci!JEEVES.UCSD.EDU!scronin From: scronin@JEEVES.UCSD.EDU (STEPHEN CRONIN) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Counting GFP colonies Date: 8 Jan 1997 10:20:08 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 21 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <32D14DE4.6D81@umich.edu> NNTP-Posting-Host: net.bio.net On 6 Jan 1997, Paul Boucher wrote: > I am interested in counting colonies that stably express GFP for > cytotoxicity assessment studies. I want to be able to count colonies > that lack and express GFP. Is there an easier way of doing this than > counting the colonies under a fluorescent microscope? Perhaps some sort > of fluorescent dish scanner. I would appreciate any input. Thank-you. > Laura Zerbe Rubsam > I have been able to look at the fluorescence of yeast colonies with high levels of s65t gfp on petri plates using a slide projector and an optical filter for illumination. Stephen Cronin scronin@jeeves.ucsd.edu From owner-fluorescent@net.bio.net Wed Jan 08 22:00:00 1997 Path: biosci!SCRIPPS.EDU!reichel From: reichel@SCRIPPS.EDU (Christoph Reichel) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Exciting with slide projector Date: 9 Jan 1997 11:27:05 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 34 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >I have been able to look at the fluorescence of yeast colonies with >high levels of s65t gfp on petri plates using a slide projector and an >optical filter for illumination. >Stephen Cronin >scronin@jeeves.ucsd.edu Dear Stephen, could you please give some details about the slide projector and Filters you used, because I think there will a lot of people be interested to set up a system like this for a lot of different purposes - not only for counting E. coli colonies, but maybe for screening plants for GFP expression..... So could you please tell me what kind of filters you used (spectra, company name) and whether you used filters for excitation and emission detection or only for emission. Thank you very much, Christoph Christoph Reichel The Scripps Research Institute Dept. Cell Biology, BCC 206 10550 North Torrey Pines Rd. La Jolla, CA 92037 U.S.A. phone: (619) 784-2918 fax: (619) 784-2994 From owner-fluorescent@net.bio.net Thu Jan 09 22:00:00 1997 Path: biosci!SCRIPPS.EDU!reichel From: reichel@SCRIPPS.EDU (Christoph Reichel) Newsgroups: bionet.molbio.proteins.fluorescent Subject: slide projector Date: 10 Jan 1997 09:43:47 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 42 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net As I guessed there is a high interest in the issue of using a slide projector (or something similiar simple) for the excitation of GFP. Since I have been asked by several interested colleques, whether I have received answer on my questions concerning excitation with a slide projector, I post this answer to the newsgroup: > >Dear Christoph Reichel; > >I've used a slide projector with a blue glass filter. In general, any >filter that cuts off before 450nm should work. Just rig is to be the same >size as a slide. > >I've found that a blacklight (bought at Spencer's in the mall) is far >superior to a slide projector for one major reason. Slide projectors use >incandescent bulbs. These bulbs don't emit much blue light and almost no >UV. Fluorescent lighting is not limited in this respect. These bulbs are >pre-filtered and work quite well for bacterial colonies. > >Seth > >Seth Jon Davis sjdavis1@students.wisc.edu >UW-Madison Horticulture Lab (608) 262-1622 >Madison, Wisconsin 53703 FAX (608) 262-4743 >http://www.wisc.edu/genetics/ > Any further suggestions will be highly appreciated, Christoph Christoph Reichel The Scripps Research Institute Dept. Cell Biology, BCC 206 10550 North Torrey Pines Rd. La Jolla, CA 92037 U.S.A. phone: (619) 784-2918 fax: (619) 784-2994 From owner-fluorescent@net.bio.net Fri Jan 10 22:00:00 1997 Path: biosci!internet!biosci!not-for-mail From: biohelp (BIOSCI Administrator) Newsgroups: bionet.molbio.proteins.fluorescent Subject: BIOSCI/bionet miniFAQ & Fundraiser Date: 11 Jan 1997 02:00:21 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 239 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701111000.CAA21929@net.bio.net> NNTP-Posting-Host: net.bio.net (LAST REVISION: 30-JUL-95) This BIOSCI "miniFAQ" is designed to answer the questions that come up the *most frequently*. The main BIOSCI FAQ (Frequently Asked Questions) is accessible on the World Wide Web at URL http://www.bio.net/. If you can not find an answer to your question in this or other documentation, the BIOSCI technical support staff answers e-mail queries sent to biosci-help@net.bio.net We can only answer questions about the use of the newsgroups and mailing lists. We unfortunately do not have the staff to do Internet information searches or answer scientific questions. Please post those to the appropriate BIOSCI/bionet newsgroups. Contents: -------- 0) BIOSCI NEEDS YOUR SUPPORT!! 1) Using the WWW to access the BIOSCI/bionet newsgroups. 2) What to do about "spams," i.e., junk mail, ads, etc. 3) Examples of subscribing and unsubscribing to the mailing lists. 4) The BIOSCI user address and research interest directory. 0) BIOSCI NEEDS YOUR SUPPORT!! ------------------------------ BIOSCI's government funding has been expended, and we are now operating solely from advertising revenue that we have raised from our Web site at http://www.bio.net/. We need just a few minutes of your time to help us serve you. You can do two important things which will take very little time for you individually and will immensely help us continue to help you. First, please use our WWW system at http://www.bio.net/ to access the archives. You can post or reply to messages via your Web browser as described in item #1 below. Your usage helps attract sponsors. If you contact any of our sponsors, please be sure to thank them for supporting BIOSCI. It is critical for them to get this feedback if they are to continue their sponsorship for the long term. Second, if you work for a company or organization that provides products or services of interest to the biology community, please pass this message on to your marketing or marketing communications department or other appropriate group. Please ask them to help support BIOSCI by sponsoring our Web site and explain the uses and benefits of the system to the biology community. If they are interested, they can then contact us for further information at our tech support address, biosci-help@net.bio.net. 1) Using the WWW to access the BIOSCI/bionet newsgroups. -------------------------------------------------------- As of 10 December 1995, all BIOSCI/bionet full newsgroups are accessible through the World Wide Web (WWW) at URL http://www.bio.net. One can read and reply publicly or privately to both recent postings and archived messages through one's Web browser if it is configured properly to send e-mail. Each newsgroup is equipped with its own WAIS index. The main BIOSCI home page also has access to the BIO-JOURNALS Table of Contents database WAIS index and the BIOSCI user address database described in another item further below. 2) What to do about "spams," i.e., junk mail, ads, etc. ------------------------------------------------------- BIOSCI is a set of parallel USENET newsgroups (the "bionet" groups), mailing lists, and a hypermail archive at URL http://www.bio.net/. The same postings are distributed on all media (except for a small number of mailing-list-only groups at net.bio.net). Unfortunately it is becoming a despicable practice on the Internet (by a few people out to make a fast buck) to do automated mass postings to thousands of newsgroups and mailing lists. These attempts to grab free advertising are refered to as "spams" in the usual, somewhat boneheaded, net terminology. USENET is more susceptible to this practice, and many spams originate on the USENET groups and then are passed on to the mailing lists. However, spammers also get lists of mailing addresses and hit these too, so neither medium is immune. What should you do personally if you get junk mail? --------------------------------------------------- Just delete it and move on without reading it further. Filing a protest is becoming increasingly useless because spammers are often disguising the addresses where the messages are sent from. Unless you really understand Internet mail systems, your attempt at protest by sending replies to the message will often end up being sent to the address of an innocent person that the spammer is victimizing. What can BIOSCI/bionet do to protect its newsgroups? ---------------------------------------------------- The only solution currently available is to moderate the newsgroup. If this newsgroup is already moderated, then you are in good shape. Moderation protects the USENET distribution from about 95% of the spams that are being sent to date and protects the mailing lists completely. 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With close to 100 newsgroups to run, the BIOSCI staff has to rely on the discussion leaders of each newsgroup to report problems directly to us at biosci-help@net.bio.net. We will moderate any of our newsgroups if the discussion leader tells us that the readership of the group wishes to do so and if a moderator is willing to do the work. For most BIOSCI/bionet groups, this entails only a few minutes of work each day. Moderating a newsgroup will resolve probably 95% of the junk postings on the USENET distribution. Unfortunately there are easy ways for determined spammers to override the moderation mechanism on USENET, but we can protect our e-mail subscribers from unwanted postings if the newsgroup is moderated. You can also access our newsgroups over the WWW at URL http://www.bio.net. While this Web interface will not stop spammers from trying to post to the groups, this will give you yet another way, besides using USENET news, to keep the junk out of your personal mail files. For those of you with local USENET news systems, the Web interface will also give you faster access to new newsgroups and recent postings. 3) Examples of subscribing and unsubscribing to the mailing lists. ------------------------------------------------------------------ PLEASE NOTE: The BIOSCI management does NOT act on subscription/unsubscription requests that are posted improperly to the newsgroups and mailing lists. People who do this only bother everyone on the lists to no avail. Please be sure to follow the proper procedures below. Gory details are in the BIOSCI Information sheets on the Web at http://www.bio.net. Below we give an example utilizing the METHODS-AND-REAGENTS list at both of our two BIOSCI sites: Users in the Americas and Pacific Rim countries who use the BIOSCI ------------------------------------------------------------------ node at computer net.bio.net: ---------------------------- A) Determine the "listname" which is the <=8 character mail address ^^^^^^^^^^^^^ for the group. These can be found in the BIOSCI Info. Sheet. For the METHODS-AND-REAGENTS group the mailing address is methods@net.bio.net. The listname is the portion of the address to the left of the @ sign, i.e., "methods". The listname is used with the "subscribe" and "unsubscribe" commands illustrated below. B) Mail all commands in the body of a mail message addressed to biosci-server@net.bio.net. Do NOT send commands to the newsgroup posting addresses! Leave the Subject: line blank, any text on it will be ignored. 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For the METHODS-AND-REAGENTS list the USENET newsgroup name is bionet.molbio.methds-reagnts, thus the appropriate commands are sub bionet-news.bionet.molbio.methds-reagnts unsub bionet-news.bionet.molbio.methds-reagnts These commands are included in a message addressed to mxt@dl.ac.uk, NOT to the newsgroup mailing addresses. As usual, include the text in the body of the message as text on the Subject: line is ignored. To unsubscribe from all the lists at the UK node, use unsub bionet-news Please note that if the address in the list is different than the one in your mail message header, you will not be able to unsubscribe by this method. If you have problems, please mail biosci@daresbury.ac.uk. 4) The BIOSCI user address and research interest directory. ----------------------------------------------------------- Please take this opportunity to add your name, address, and research interest information to the BIOSCI User Address Database if you have not already done so. You can fill out the address form directly through our Web page at URL http://www.bio.net/adrform.html. The address database is reindexed nightly for WWW access (the URL is http://www.bio.net/). If you are not directly on the Internet but can reach it by e-mail, please use our waismail server to access the user directory. waismail use is described above. You can also request a user address form by e-mail from biosci-help@net.bio.net. Please check your database entry from time-to-time to see if your address information is still up-to-date. Because of our limited personnel resources, we ask that you resubmit a *complete* form to revise your entry; we only replace complete entries and do not have resources to edit old forms. Sincerely, Dave Kristofferson BIOSCI/bionet Manager biosci-help@net.bio.net From owner-fluorescent@net.bio.net Fri Jan 10 22:00:00 1997 Path: biosci!JEEVES.UCSD.EDU!scronin From: scronin@JEEVES.UCSD.EDU (STEPHEN CRONIN) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: Exciting with slide projector Date: 11 Jan 1997 11:44:13 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 25 Sender: daemon@net.bio.net Distribution: world Message-ID: References: NNTP-Posting-Host: net.bio.net Dear Christoph, Here is some information about my illumination techniques and apparatus. The yeast I am screening have an S65T version of GFP which has an excitation wavelength of 488nm and an emission wavelength of 512nm. For illumination I use a Kodak 4400 slide projector with an optical filter in the slide slot. There is nothing special about the slide projector that I know of; I think that any one with a decent bulb will work. The filter is a 488DF22 narrow bandpass filter from Omega Optical (802/254-2690) matched to the excitation wavelength of the S65T gfp (488nm). The filter I have is a 50mmX50mm square, the size of a slide. It has low band blocking on it so that it will not transmit red light. Since the 488nm blue illuminating light is visible, a filter is needed to view the much less intense 512nm green fluorescence. I use two Kodak No.12 gelatin filters taped to a UV face sheild. The UV face sheild is not needed but it is a convinient way to hold the filters. THis is definitely not the most sensitive way to detect fluorescence. I can detect much weaker fluorescence at the cell level using a fluorescence mircroscope or FACS, but I does work for my purposes. I hope this information will help. Stephen Cronin scronin@jeeves.ucsd.edu From owner-fluorescent@net.bio.net Sun Jan 12 22:00:00 1997 Path: biosci!daresbury!not-for-mail From: Stephane.Pasteau@pasteur-lille.fr (Stephane Pasteau) Newsgroups: bionet.molbio.proteins.fluorescent Subject: stability in bacteria Date: 13 Jan 1997 16:16:49 -0000 Lines: 18 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5bdn5h$k3i@mserv1.dl.ac.uk> Original-To: fluorpro@dl.ac.uk Has anybody informations about stability of the GFP in bacteria cells (E. coli for exemple). How many days (or hours) it persists in cells after the expression induction? In a mediun unsupplemented with ampicillin, during how many time bacterial cells possessing the GFP plasmid are able to express the GFP? Thanks for the informations you could give me. Dr Stephane PASTEAU Institut Pasteur de Lille Service de Recherche et D=E9veloppement Domaine du CERTIA 369, rue Jules Guesde - B.P. 39 59651 VILLENEUVE D'ASQ cedex tel. : (33) 03-20-43-86-67 fax. : (33) 03-20-43-86-66 e.mail : Stephane.Pasteau@pasteur-lille.fr From owner-fluorescent@net.bio.net Mon Jan 13 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!sn.no!nntp.uio.no!news-feed.inet.tele.dk!enews.sgi.com!news.sgi.com!news.bbnplanet.com!su-news-hub1.bbnplanet.com!newsxfer3.itd.umich.edu!howland.erols.net!vixen.cso.uiuc.edu!ais.net!noc.van.hookup.net!nic.mtl.hookup.net!rcogate.rco.qc.ca!chinook.generation.net!news@generation.net From: "Marja E. Coady" Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: slide projector Date: Tue, 14 Jan 1997 19:43:56 -0500 Organization: génération.NET Lines: 47 Message-ID: <32DC284C.815@generation.net> References: NNTP-Posting-Host: portD10.Generation.NET Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Christoph Reichel wrote: > > As I guessed there is a high interest in the issue of using a slide > projector (or something similiar simple) for the excitation of GFP. Since I > have been asked by several interested colleques, whether I have received > answer on my questions concerning excitation with a slide projector, I post > this answer to the newsgroup: > I'd like to throw in my two cents on the use of slide projectors for visualizing GFP. We've done this for quite a while and I've a couple of small suggestions to make. One is to consider not only which excitation wavelength to choose for the filter attached to the slide projector, but also the size of the bandpass. We use GFP when we inject DNA into Xenopus oocytes, which are about 1 mm in diameter and thus far too large for use with a microscope. We tried a few filters from Andover Technologies (Salem, MA) and decided to go with the 400 nm filter with a wavelength half-maximum of 40 nm (Cat #400FS40-50). In our hands, this permitted easy visualization of regular GFP as well as sufficient illumination that we could make out the cells that were not fluorescent, making it easy to separate the two. For some uses, this may be essential. In any event, filters can be purchased with half-maxima of 10 or 70 nm, I believe. Another factor is the emission filter used. I suspect (but am not sure) that Kodak Wratten filters are similar to the type that we purchased (Lee transparent green cellophane photography gel (Green #122)). In any case, the Lee gels can be purchased at most good photography shops and are surprisingly inexpensive to anyone who purchases scientific equipment. This green gel is ideal for visualizing GFP and is available in different sizes, to be attached to visors or glasses as desired. Do not let any industrious individual try to clean them with alcohol!!!!! :-< Finally, it is useful to buy some inexpensive black cloth to block light from the slide projector from interfering with visualizing the GFP. It is strongly advised that you put it up yourself and affix some kind of sign so that nobody gets the bright idea to try to wrap it around the slide projector, blocking the ventilation. Yes, it does happen. Mike (no affiliation with the companies mentioned) Coady GFTM Universite de Montreal From owner-fluorescent@net.bio.net Tue Jan 14 22:00:00 1997 Path: biosci!rutgers!csn!nntp-xfer-1.csn.net!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!worldnet.att.net!news.u.washington.edu!root From: "Robert E. Hughes" Newsgroups: bionet.molbio.proteins.fluorescent Subject: Bleaching of GFP Date: 15 Jan 1997 22:31:35 GMT Organization: Howard Hughes Medical Institute Lines: 12 Message-ID: <5bjls7$ecg@nntp3.u.washington.edu> NNTP-Posting-Host: 128.95.12.37 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins.fluorescent#5bdn5h$k3i@mserv1.dl.ac.uk Does anyone have any infromation on how to minimize bleaching of GFP expressed in Yeast cells? I'm trying to do confocal fluorescence microscopy on Yeast and have been having a problem with GFP fading. Any comments or references would be greatly appreciated. Robert E. Hughes University of Washington Seattle, WA rehughes@u.washington.edu From owner-fluorescent@net.bio.net Tue Jan 14 22:00:00 1997 Path: biosci!agate!howland.erols.net!www.nntp.primenet.com!nntp.primenet.com!news.primenet.com!news.primenet.com!not-for-mail From: "Tankertom" Newsgroups: bionet.molbio.proteins.fluorescent Subject: Best clone for vertebrate expression? Date: 15 Jan 1997 23:02:02 -0700 Organization: Primenet Services for the Internet Lines: 7 Message-ID: <01bc0372$f4a76f20$be2a44c6@primenet.primenet.com> X-Posted-By: @198.68.42.190 (mccreer) X-Newsreader: Microsoft Internet News 4.70.1155 We are interested in using GFP as a marker both in human cell lines and in vivo. What is the best mutant currently available. Could anyone recommend a source for this mutant. Thanks, Tom McCreery From owner-fluorescent@net.bio.net Tue Jan 14 22:00:00 1997 Path: biosci!botanik.biologie.uni-muenchen.de!leeder From: leeder@botanik.biologie.uni-muenchen.de (Stephanie Leeder) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Help: GFP expression in Chlamydomonas Date: 15 Jan 1997 05:46:52 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.16.19970115144507.11f77f1e@odin.botanik.biologie.uni-muenchen.de> NNTP-Posting-Host: net.bio.net Hello our workgroup is starting a new project: trying to express GFP in Chlamydomonas Because we are just starting it would be a great help if we could get any kind of information about: 1) groups who are working with the same subject? 2) are there already papers or other articles availible about expression experiments with GFP in Chlamydomonas? 3) which would be the best GFP-mutant to start with? I have some experience with the S65T-mutant, but are there any mutants which are G/C richer and whould be therefore better for Chlamydomonas? Any kind of addresses and information would be a great help. Thanks very much: the biophysik group from Prof. Uhl, University of Munich From owner-fluorescent@net.bio.net Tue Jan 14 22:00:00 1997 Path: biosci!BUSTOFF.BWH.HARVARD.EDU!csweber From: csweber@BUSTOFF.BWH.HARVARD.EDU ("Dr. C.B. Schmidt-Weber") Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in retroviral vectors Date: 15 Jan 1997 08:12:38 -0800 Organization: Brigham and Women's Hospital, Dept. of Immunology, Immunology Research Division, Harvard Medical School, Boston, USA Lines: 5 Sender: daemon@net.bio.net Distribution: world Message-ID: <32DD1027.779A@bustoff.bwh.harvard.edu> Reply-To: csweber@bustoff.bwh.harvard.edu NNTP-Posting-Host: net.bio.net We are interested to express GFP in T cells for promotor analysis. We would like to transfect T cells with retrovirally encoded GFP under konstitutive expression. Carsten From owner-fluorescent@net.bio.net Wed Jan 15 22:00:00 1997 Path: biosci!agate!howland.erols.net!worldnet.att.net!news.u.washington.edu!root From: "Robert E. Hughes" Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in E.coli Date: 17 Jan 1997 03:01:16 GMT Organization: Howard Hughes Medical Institute Lines: 10 Message-ID: <5bmq1s$o8e@nntp3.u.washington.edu> NNTP-Posting-Host: 128.95.12.37 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) X-URL: news:bionet.molbio.proteins.fluorescent Ive been expressing GFP fusions in e.coli and have been seeing bright subcellular localization. I'm concern that these may be inclusion bodies. Does anyone have any experience looking at GFP fusions in coli? Has anyone observed fluorescent inclusions bodies? I'd appreciate hearing from anyone with observations in this area. Thanks. Robert E. Hughes University of Washington From owner-fluorescent@net.bio.net Wed Jan 15 22:00:00 1997 Path: biosci!SCRIPPS.EDU!reichel From: reichel@SCRIPPS.EDU (Christoph Reichel) Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: GFP in E.coli Date: 16 Jan 1997 20:02:24 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 24 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net >Ive been expressing GFP fusions in e.coli and have been seeing bright >subcellular localization. I'm concern that these may be inclusion >bodies. Does anyone have any experience looking at GFP fusions in coli? >Has anyone observed fluorescent inclusions bodies? I'd appreciate >hearing from anyone with observations in this area. Thanks. > >Robert E. Hughes >University of Washington Dear Robert, my understanding is that GFP expressed in inclusion bodies (if as fusion or alone) is non-fluorescent. When I initially expressed GFP from a T7 promoter in BL21 cells I got very high levels of GFP, but all of it was expressed as insoluble, non-fluorescent inclusion bodies. Changing culture conditions (temperature, media etc.) did not help at all. In the end I used a different (weaker) promoter (lac promoter from Bluescript vector) and got sufficient levels of functional, soluable protein (at 37 degree Celsius). Hope that helps you, Christoph From owner-fluorescent@net.bio.net Wed Jan 15 22:00:00 1997 Path: biosci!GOOFY.ZDV.UNI-MAINZ.DE!wirts000 From: wirts000@GOOFY.ZDV.UNI-MAINZ.DE (Stefan Wirtz) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in frozen sections Date: 16 Jan 1997 02:20:32 -0800 Organization: Uniklinik Mainz Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <32DE0F42.62CF@mail.uni-mainz.de> Reply-To: wirts000@mail.uni_mainz.de NNTP-Posting-Host: net.bio.net I would like to detect GFP in tissue sections (exspecially frozen sections of the gut). Is GFP stable in frozen sections ? Would autoflourescence be a problem ? Thank you for any comments. Best regards, Stefan Wirtz I. Medical Clinic University of Mainz, Germany E-mail : wirts000@mail.uni-mainz.de From owner-fluorescent@net.bio.net Wed Jan 15 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!sn.no!nntp.uio.no!newsfeeds.sol.net!hammer.uoregon.edu!news.icm.edu.pl!news.nask.pl!01-newsfeed.univie.ac.at!03-newsfeed.univie.ac.at!mail.boku.ac.at!news From: iam@mail.boku.ac.at (Birgit Dalheimer) Newsgroups: bionet.molbio.proteins.fluorescent Subject: gfp in plants Date: 16 Jan 1997 13:53:16 GMT Organization: IAM Institute of Applied Microbiology Lines: 5 Message-ID: <5blbsc$rcg@mail.boku.ac.at> NNTP-Posting-Host: 141.244.135.61 X-Newsreader: WinVN version 0.82 I m trying to find information about the expression of GFP in plants. What I m interested in is the stable integration of the gfp-DNA into the plant genome and the regeneration capacity of undifferentiated cells expressing GFP to develop into fully trans formed plants. Has anyone got any experience with that or know something about it? Thank you, Birgit Dalheimer From owner-fluorescent@net.bio.net Mon Jan 20 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!nntp.uio.no!newsfeeds.sol.net!news-xfer.netaxs.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!usenet.uchc.edu!panda.uchc.edu!skwon From: Sunjong Kwon Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: Tue, 21 Jan 1997 14:18:31 -0500 Organization: Univ of CT Health Center Lines: 11 Message-ID: NNTP-Posting-Host: panda.uchc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII I want to subscribe bionet.molbio.proteins.fluorescent. Sunjong Kwon Department of Biochemistry University of Connecticut Health Center Farmington, CT 06030 Tel:860-679-2106 Fax:860-679-3408 e-mail:skwon@panda.uchc.edu From owner-fluorescent@net.bio.net Tue Jan 21 22:00:00 1997 Path: biosci!MED.UNC.EDU!lemaster From: lemaster@MED.UNC.EDU ("John J. Lemasters") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Subscribe Date: 22 Jan 1997 11:45:24 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 2 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701221945.OAA11845@garner.med.unc.edu> NNTP-Posting-Host: net.bio.net Subscribe From owner-fluorescent@net.bio.net Tue Jan 21 22:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cam-news-hub1.bbnplanet.com!usenet.uchc.edu!panda.uchc.edu!skwon From: Sunjong Kwon Newsgroups: bionet.molbio.proteins.fluorescent Subject: subscribe Date: Wed, 22 Jan 1997 11:56:15 -0500 Organization: Univ of CT Health Center Lines: 4 Message-ID: NNTP-Posting-Host: panda.uchc.edu Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII subscribe fluorpro end From owner-fluorescent@net.bio.net Tue Jan 21 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!online.no!sn.no!hermod.uio.no!nntp.uio.no!news-feed.inet.tele.dk!enews.sgi.com!news.sgi.com!howland.erols.net!news.sprintlink.net!news-peer.sprintlink.net!news-pull.sprintlink.net!news.sprintlink.net!news-dc-9.sprintlink.net!newsjunkie.ans.net!newsfeeds.ans.net!news.lava.net!nntp.reed.edu!sun.lclark.edu!sun.lclark.edu!kuhn From: kuhn@sun.lclark.edu (Sam Kuhn) Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP antibody and EGFP? Date: 22 Jan 1997 01:36:55 GMT Organization: Lewis & Clark College, Portland OR Lines: 9 Message-ID: <5c3qvn$438@sun.lclark.edu> NNTP-Posting-Host: sun.lclark.edu X-Newsreader: TIN [version 1.2 PL2] I am looking for other people who have used Clontech's GFP antibody (polyclonal) against EGFP, or EGFP fusion proteins. I would be especially interested in experience involving the use the antibody in westerns. Thank you, - Sam Kuhn Lewis & Clark College From owner-fluorescent@net.bio.net Fri Jan 24 22:00:00 1997 Path: biosci!BIOSUN.HARVARD.EDU!pdanese From: pdanese@BIOSUN.HARVARD.EDU (Paul N. Danese) Newsgroups: bionet.molbio.proteins.fluorescent Subject: "punctate" staining of GFP in E. coli Date: 25 Jan 1997 20:02:41 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 28 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I've been expressing a cytoplasmic GFP fusion protein in E. coli. I had assumed that this protein would be found evenly distributed throughout the E. coli cytoplasm. However, I observe several "dots" of fluorescence (2-11 dots) per cell, and I'm concerned that I'm concentrating fluorescent protein into inclusion bodies. I've typically read that GFP (and it's derivative fusion proteins) is(are) not fluorescent in inclusion bodies. However, has anyone ever observed FLUORESCENT GFP in inclusion bodies? Has anyone ever expected to see even distribution of GFP within a cell and observed localized fluorescence instead? Thanks in advance for your help. Sincerely, Paul N. Danese Department of Molecular and Cellular Biology 16 Divinity Avenue Harvard University Cambridge, MA 02138 email: pdanese@biosun.harvard.edu phone: 617-495-4217 FAX: 617-496-1114 From owner-fluorescent@net.bio.net Sat Jan 25 22:00:00 1997 Path: biosci!MED.UNC.EDU!lemaster From: lemaster@MED.UNC.EDU ("John J. Lemasters") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Carolina Workshop on: LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES Date: 26 Jan 1997 10:59:02 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 118 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701261859.NAA07986@garner.med.unc.edu> NNTP-Posting-Host: net.bio.net ------------------------------------------------------------------- COURSE ANNOUNCEMENT Carolina Workshop on: LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES June 1-6, 1997 University of North Carolina at Chapel Hill Instructors: John J. Lemasters Edward D. Salmon Brian Herman ------------------------------------------------------------------- LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES is an introduction to applications of light microscopy. Students will have opportunities for extensive hands-on experience with state-of-the-art equipment for optical imaging, digital imaging processing, fluorescence microscopy and confocal microscopy guided by experienced academic and commercial staff. The course is divided into three major sections with lectures and laboratory exercises on: 1) geometric and wave optics of image formation, microscope alignment, phase contrast and reflection interference contrast microscopy; 2) video imaging, including contrast enhancement by analog and digital image processing, fluorescence microscopy, image detectors, fluorescent probes, ion imaging, and green fluorescent protein; and 3) laser scanning confocal microscopy emphasizing live cell imaging and 3-dimensional image reconstruction. Students are encouraged to bring their own specimens for analysis. The workshop on LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES will cover basic concepts of light microscopy and introduce several advanced techniques relevant to modern cell and molecular biology. A commercial staff representing leading microscopic manufacturers will make available for student use the latest and most advanced instrumentation for light microscopy, image detection and computerized image analysis. ------------------------------------------------------------------- APPLICATION FORM Carolina Workshop on LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES Name: Position: Address: Telephone: Fax: E-mail: Please return this form along with a brief letter describing your research interests and a curriculum vitae. Applicants should contact the program as soon as possible. Full consideration will be given to applications received by April 18, 1997. Send application to: Dr. Wayne Litaker, Director of Workshops University of North Carolina at Chapel Hill Program in Molecular Biology & Biotechnology CB# 7100, 442 Taylor Hall Chapel Hill, North Carolina 27599-7100 Tel: (919) 966-1730 Fax: (919) 966-6821 e-mail: litaker@unc.med.edu ------------------------------------------------------------------- About Carolina Workshops: CAROLINA WORKSHOPS are intensive hands-on laboratory courses designed to teach cutting edge methods in molecular biology and biotechnology. Several courses on different topics in molecular biology and biotechnology are offered each year by the Program in Molecular Biology & Biotechnology at the University of North Carolina at Chapel Hill. Most participants in the Carolina Workshops already hold M.D. or Ph.D. degrees or are advanced pre-doctoral students. The courses are designed for novice students as well as for individuals with prior experience. All students benefit from in-depth interaction with instructors. ------------------------------------------------------------------- About the Instructors: John J. Lemasters, M.D., Ph.D. (Course Director): Dr. Lemasters is Professor and Director of Confocal Imaging in the Department of Cell Biology & Anatomy. Dr. Lemasters' research interests center on toxic and hypoxic injury, liver preservation for transplantation and mitochondrial calcium homeostasis, using confocal microscopy to monitor ions, membrane potentials, cell volumes, oxygen radicals and other parameters in single living cells. Brian Herman, Ph.D: Dr. Herman is Professor and Co-Director of the Digitized Video Microscopy Facility in the Department of Cell Biology & Anatomy. Dr. Herman's research addresses the role of calcium, tumor suppressor genes, and anti-apoptotic proteins on regulation of cell growth and cell death using techniques of digital ion imaging, resonance energy transfer, confocal microscopy and fluorescence life time imaging. Edward (Ted) D. Salmon, Ph.D: Dr. Salmon is a Professor in the Department of Biology whose interests are cell biology, cell motility, microtubules and mechanisms of mitosis and cell division. Dr. Salmon's research applies high resolution video and digital imaging microscopy towards understanding the molecular mechanisms governing the assembly of spindle microtubules and the segregation of chromosomes during mitosis. ------------------------------------------------------------------ Carolina Workshop on: LIGHT MICROSCOPY FOR THE BIOMEDICAL SCIENCES June 1-6, 1997 University of North Carolina at Chapel Hill Instructors: John J. Lemasters Edward D. Salmon Brian Herman ------------------------------------------------------------------- From owner-fluorescent@net.bio.net Sun Jan 26 22:00:00 1997 Message-ID: <32ECAF1B.645B@chuv.hospvd.ch> Date: Mon, 27 Jan 1997 14:35:23 +0100 From: Anne PARRICAL Organization: CHUV X-Mailer: Mozilla 2.02 (Win95; I) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins.fluorescent Subject: test de message Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit NNTP-Posting-Host: pc1bh08407.hospvd.ch Lines: 2 Path: biosci!rutgers!news.sgi.com!howland.erols.net!EU.net!Portugal.EU.net!news.rccn.net!swidir.switch.ch!cisun2000.unil.ch!pc1bh08407.hospvd.ch ceci est un test From owner-fluorescent@net.bio.net Sun Jan 26 22:00:00 1997 From: Barbara Loeliger Organization: CHUV X-Mailer: Mozilla 1.1N (Windows; I; 16bit) MIME-Version: 1.0 Newsgroups: bionet.molbio.proteins.fluorescent Subject: Quenching the GFP fluorescence Content-Transfer-Encoding: 7bit Content-Type: text/plain; charset=us-ascii NNTP-Posting-Host: pc1bh19123.hospvd.ch Message-ID: <32ecc393.0@cisun2000.unil.ch> Date: 27 Jan 97 15:02:43 GMT Lines: 5 Path: biosci!rutgers!news.sgi.com!news-peer.gsl.net!news.gsl.net!EU.net!Portugal.EU.net!news.rccn.net!swidir.switch.ch!cisun2000.unil.ch!pc1bh19123.hospvd.ch Does anybody know something how GFP fluorescence could be quenchend in vivo? I am working with GFP marked bacteria and looking for a powerfull quencher in a in vivo assay.Can anyone give me some advices? Many thanks, Barbara From owner-fluorescent@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!daresbury!nntp-trd.UNINETT.no!sn.no!www.nntp.primenet.com!nntp.primenet.com!su-news-hub1.bbnplanet.com!news.bbnplanet.com!cpk-news-hub1.bbnplanet.com!worldnet.att.net!news.u.washington.edu!root From: "Robert E. Hughes" Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: "punctate" staining of GFP in E. coli Date: 29 Jan 1997 02:46:14 GMT Organization: Howard Hughes Medical Institute Lines: 14 Message-ID: <5cmdlm$fq0@nntp3.u.washington.edu> References: NNTP-Posting-Host: 128.95.12.37 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 1.1N (Macintosh; I; 68K) To: pdanese@BIOSUN.HARVARD.EDU X-URL: news:v01510102af10888bf09b@[140.247.92.142] Paul: Regarding the fluorescent inclusion body issue, I've have several messages stating that it's generally accepted that inclusion bodies do not fluoresce. Since you are the first person who says he's read this, I'm wondering if you could direct me to some references that address this question. Thanks very much Robert E. Hughes Howard Hughes Medical Institute University of Washington, Seattle WA rehughes@u.washington.edu From owner-fluorescent@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!CMB.BIOSCI.WAYNE.EDU!hivlab From: hivlab@CMB.BIOSCI.WAYNE.EDU ("A. S. Goustin Lab") Newsgroups: bionet.molbio.proteins.fluorescent Subject: Re: test de message Date: 28 Jan 1997 14:17:10 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: References: <32ECAF1B.645B@chuv.hospvd.ch> NNTP-Posting-Host: net.bio.net succee! il marche bien On Mon, 27 Jan 1997, Anne PARRICAL wrote: > ceci est un test > > From owner-fluorescent@net.bio.net Mon Jan 27 22:00:00 1997 Path: biosci!agate!howland.erols.net!rill.news.pipex.net!pipex!warwick!lyra.csx.cam.ac.uk!daresbury!not-for-mail From: Xavier Gansel Newsgroups: bionet.molbio.proteins.fluorescent Subject: GFP in plants Date: 28 Jan 1997 17:36:53 -0000 Lines: 73 Sender: lpddist@mserv1.dl.ac.uk Distribution: bionet Message-ID: <5cldfl$64h@mserv1.dl.ac.uk> Original-To: fluorpro@dl.ac.uk Dear netters, A few weeks ago I posted the following question to this news-group as well as to people known, from the bibliography, to be working on GFP in plants. >Here is a mail to ask for advices on the use of GFP in A. thaliana >I wish to use GFP as a reporter gene to follow the regulation >(activation/repression, tissue and cell expression, etc. ....) of a >promoter. >So, I want to get transgenic A.t. plants carrying a GFP under the control >of this promoter. > >From the bibliography, it appears that their is a growing number of "plant >optimised" GFP. The optimisations beeing on different aspects. Some of them >are the optimisation of codon usage, the removal of a cryptic A.t. intron, >the addition of a plant intron, mutations that shift light emission or >enhance thermoresistance, anchoring in the ER to reduce toxicity of nuclear >GFP accmlulation (Chui, 1996, Curr. Biol., 6, 325-330 // Davis, Weeds >World Volume 3(ii) // Haseloff, TIG, 1951, 11, 328-329 // Haseloff, poster >S44, 7th International Conference on Arabidopsis Research // Pang, Plant >Physiol., 1996, 112, 893-900). >But all those mutations/improvements are not present on the same plant-GFP. > >My questions are the following : >1/ on today statut, which version of plant improved GFP would you recommend >to be use as the best reporter system in A.t. ? (the promoter to be studied >may be weak and so require a very sensitive reporter gene to detect it) >2/ were could I get this "super" plant-GFP ? Up to today I obtained only the two following answers. Of course I thank a lot Jim Haseloff and Seth J. Davis for the kind informations they provided. Cheers, Xavier. -------------------------------------------------------------------------------- 1// From: sjdavis1@students.wisc.edu (Seth J. Davis) Dear Xavier Gansel, 1) What is the best GFP for promoter studies in Arabidopsis thaliana? 2) Where do you get it? The first question is difficult to answer, as none of the plant-use GFPs have been fully characterized. In Arabidopsis I believe several factors should be included in any choice. First, the optimized codon use should be included, and a soluble derivative should be used. Under this criteria, there are GFPs generated from two labs: the Vierstra Lab and the Haseloff Lab. It is not clear who's GFP is "better." But if your going to use the GFP for promoter studies, and have the capacity for fluorescensce microscopy, I would choose either psmRSGFP or mGFP5. These two GFPs have not been compared sized by side in promoter studies, but it is being done here at the UW-Madison (by several labs). As for the second question, psmRSGFP can be obtained from the Arabidopsis Stock Center at the Ohio State University. Haseloff's email is jph@alf1.mrc-lmb.cam.ac.uk. You should contact him for mGFP5. Seth -------------------------------------------------------------------------------- 2// From: jph@mrc-lmb.cam.ac.uk (Jim Haseloff) Hi Xavier, I'll have you sent some of our latest modified GFP - it comes with full details, and should be want you want - There are partial details in the December issue of Current Biology. ------------------------------------------------------------------------------- fluorpro@dl.ac.uk From owner-fluorescent@net.bio.net Tue Jan 28 22:00:00 1997 Path: biosci!unsw.edu.au!C.Marquis From: C.Marquis@unsw.edu.au (Chris Marquis) Newsgroups: bionet.molbio.proteins.fluorescent Subject: eGFP expression in e.coli Date: 29 Jan 1997 18:42:38 -0800 Organization: Biotechnology, The University of New South Wales Lines: 11 Sender: daemon@net.bio.net Distribution: world Message-ID: <199701300243.NAA15955@sam.comms.unsw.EDU.AU> NNTP-Posting-Host: net.bio.net dear all, has anyone tried to express any of the GFP mutants (S65T, EGFP, RSGFP) in e.coli? most of the commercial vectors seem to have codons optimised for mammalian cell expression. i would appreciate any feed back on this. thanks in advance chris marquis