Marker Gene Monthly Newsletter   

February, 2004

Volume 4, Number 2

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Bacterial Quantitation using FDG

Fluorescein di-b-D-Galactopyranoside (FDG, M0250) is a popular substrate for measuring cloned beta-Galactosidase activity in living cells.  It also has found significant utility in staining and quantifying bacteria, as well as in isolating specific strains of bacteria by fluorescence activated cell-sorting (FACS) analysis.  FDG has been found to be about 70 times more sensitive in bacterial assays than GFP.  The standard assay for quantitating the amount of b-galactosidase activity in cells, originally described by Miller for assay of bacterial cultures, involves spectrophotometric measurement of the formation of the yellow chromophore o-nitrophenol (ONP) as the hydrolytic product of the action of b-galactosidase on the colorless substrate o-nitrophenyl-b-D-galactoside (ONPG). The amount of ONP produced as a function of reaction time per volume of cell culture is divided by the optical density of the culture to generate a value of specific enzyme activity in Miller units.  Analogous methods using FDG are 2-3 orders of magnitude more sensitive for bacterial detection in a microtiterplate format than colorimetric assays. For bacterial cell permeabilization in a microplate format, 100 uL of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM b-mercaptoethanol) is dispensed into the microplate wells.  After adding 20 uL of freshly prepared 0.1% SDS and 40 uL of chloroform into wells, a 12-channel pipettor is used to transfer an aliquot of cell culture (50–100 uL). Permeabilization is accomplished by pipetting contents up and down a few times to mix. In the standard Miller method, permeabilization was achieved by vortexing, one at a time, test tubes containing the cell-chloroform/SDS mixture for 10 sec.  For more information about FDG staining and quantitation of bacteria, please see our Web site or see the references below:

• b-Galactosidase Activity in Single Differentiating Bacterial Cells." F. Russo-Marie, Roederer, M. Sager, B., Herzenberg, L., Proc. Natl. Acad. Sci. USA 90:8194 (1993).
• Nelis, H.; Van Poucke, S. Enzymatic detection of coliforms and Escherichia coli within 4 hours." Water, Air, and Soil Pollution (2000), 123(1-4): 43-52.
• Dreier, Jurg; Breitmaier, Eva B.; Gocke, Elmar; Apfel, Christian M.; Page, Malcolm G. P. Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay." Mutation Research (2002), 513(1-2): 169-182.
• Rowland B ; Purkayastha A ; Monserrat C ; Casart Y ; Takiff H ; McDonough KA (1999) “Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species” FEMS Microbiol. Lett. 179(2): 317-25.
• Kevin L. Griffith and Richard E. Wolf, Jr. (2002) “Measuring b-Galactosidase Activity in Bacteria: Cell Growth, Permeabilization, and Enzyme Assays in 96-Well Arrays”  Biochemical and Biophysical Research Communications 290: 397–402.
• Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  

lux Marker Gene used for Tracing Bacterial Growth in vivo.

The luciferase genes (luxAB) flanked by the genes involved in synthesis of its fatty aldehyde substrate (luxCDE), have been utilized to monitor cell growth in vivo.  Dr. Chris Contag and fellow workers at Stanford University and Xenogen Corporation have harnessed this marker gene system to follow the growth of bacteria inside living tissue, by utilizing a CCD low light photographic approach (Bioluminescence imaging, BLI).  The light intensity per cell is highly dependent on cellular growth resulting in a spectacular autoinduction of luminescence at high cell density. To date, this bacterial luxABCDE system has found use for monitoring bacterial infections from persistent Listeria monocyogenes infections in the murine gall bladder (see photo) or pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.  For more information about these exciting new technologies for tracking marker genes in vivo, see the references below:

• Francis K.P., Yu J., Bellinger-Kawahara C., Joh D., Hawkinson M.J., Xiao G., Purchio T.F., Caparon M.G., Lipsitch M., Contag P.R., (2001) “ Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.” Infect Immun. 69(5): 3350-3358.
• Jonathan Hardy, Kevin P. Francis, Monica DeBoer, Pauline Chu, Karine Gibbs, and Christopher H. Contag, (2004) “Extracellular Replication of Listeria monocytogenes in the Murine Gall Bladder”, Science 303(5659): 851-853.

Antibody Labeling with FITC.

Direct labeling of primary antibodies eliminates the need to use a secondary Ab for detection, providing lower background and higher sensitivity.  Marker Gene is introducing our new FITC Antibody Labeling Kit (M0955), containing a highly reactive dye, FITC, isomer I, that forms the desired dye-protein conjugates by reacting with the primary amines of proteins, in an easy two-hour protocol.  This kit produces highly green fluorescent antibodies with absorption and emission maxima of 494nm and 520nm, respectively.  These wavelengths match most fluorescence microscopes, microplate readers or FACS systems.  The kit contains enough reagents for 5 labelings, including buffers, solvents, gel-filtration chromatography media for purification and a detailed protocol for use. Please contact our technical services department for further information or to order.  Bulk discounts are available on this item! 

• Brinkley, M., (1992) “A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents.” Bioconjug Chem. 3(1): 2-13
• Haugland, R.P., (1995) “Coupling of monoclonal antibodies with fluorophores.” Methods Mol Biol. 45: 205-221
• Jung Y.S., Frank J.F., Brackett R.E., (2003) “Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes.” J Food Prot. 66: 1283.

New lacZ Vector with High Expression Efficiency.

Dr. Donald Anson and coworkers at the Department of Chemical Pathology, Women's and Children's Hospital in North Adelaide, Australia have developed a new lacZ expression vector with high expression efficiency in mammalian cells.  The coding sequence for the E. coli beta-galactosidase gene was codon-optimised and when expressed in mammalian cells resulted in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct the codon-optimised gene gave an approximately five-fold increase in apparent titre, as determined by X-Gal staining, in comparison to an analogous construct containing the native E. coli gene.  In addition, codon-optimisation resulted in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E. coli gene sequence. For more information on this new vector system, see the references below.  Look for these new vectors in kits from Marker Gene soon!

• Anson D.S., Limberis M., (2004) “An improved beta-galactosidase reporter gene.” J Biotechnol 108(1): 17-30.  
• http://biology.kenyon.edu/courses/biol09/betagal/FRAMES/
• MacGregor, G. R. & Caskey, C. T. (1989) “Construction of plasmids that express E. coli beta-galactosidase in mammalian cells.” Nucleic Acids Res. 17:2365.
• Okayama, H. & Berg, P. (1983) “A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.” Mol. Cell Biol. 3:280-289.
• MacGregor, G. R.,et al. (1987) “Histochemical staining of clonal mammalian cell lines expressing E. coli beta-galactosidase indicates heterogeneous expression of the bacterial gene.” Somat. Cell Mol. Genet. 13:253-265.
  
  

New Chemiluminescent LacZ b-Galactosidase Detection Kit.

The E. coli lacZ gene codes for an active subunit of ß-galactosidase in live cells.  Since this enzyme is generally absent in normal mammalian, yeast, some bacterial and even plant cells, it can be used as a fusion marker, and detected at very low levels.  In addition, its wide substrate specificity allows monitoring of lacZ expression (and therefore co-expressed genes or promoter efficiency) of as few as 5 copies of the ß-galactosidase protein. Although chromogenic assays of ß-galactosidase activity (i.e. X-Gal) are useful, the recent application of chemiluminescent 1,2 dioxetane substrates, which emit visible light upon enzyme catalysis, provide rapid results with very low background and high intensity signal.  Our new Chemiluminescent lacZ b-Galactosidase Detection Kit may be as much as 30X more sensitive than conventional assay techniques, in live cells.  An enhancing solution is also provided with the kit to increase light production efficiency.  The Chemiluminescent lacZ ß-Galactosidase Detection Kit (M0855) provides all the necessary reagents, buffers, substrate, and a detailed protocol for sensitive and quantitative lacZ ß-galactosidase activity assay.  Please contact our technical services department for further information, or see the references below for more details.

• “New β-Galactosidase Chemiluminescent Live Cell Assay System.” L. Tsai, B. Giri, H. R. Toben, J. J. Naleway, (2003) Mol. Biol. Cell 14: 468a.
• Chemiluminescent 1,2-dioxetanes. Brij P. Giri, PCT/US99/20590.
• Novel stabilized formulations for chemiluminescent assays. Brij P Giri, PCT/US01/02779.
• Single molecule detection of Alkaline Phosphatase enzyme using enhanced chemiluminescent from 1,2-dioxetanes and water-soluble, water-insoluble or partially water-soluble polymers. Brij P Giri, US Patent Application, 2002/0013250.
  
  

2004-2005 Catalog Will Be Available Soon.

The 2004-2005 edition of the Marker Gene catalog is in production.  Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products.  Be sure to add your name to our mailing list.  Please visit our Web site and fill out our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you.
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Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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