Download PDF by Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.): Applications of Chimeric Genes and Hybrid Proteins

By Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.)

The significantly acclaimed laboratory common for greater than 40 years, tools in Enzymology is among the such a lot hugely revered guides within the box of biochemistry. when you consider that 1955, each one quantity has been eagerly awaited, usually consulted, and praised by means of researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless correct today-truly a necessary ebook for researchers in all fields of lifestyles sciences.

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To illustrate these applications, we use examples from two intracellular bacterial pathogens: Salmonella typhimurium and Mycobacterium marinum. Green Fluorescent Protein as a Selectable Marker to Identify Differentially Expressed Genes Traditional "promoter trap" strategies have identified environmentally regulated genes by creating random gene fusions to reporter genes whose translated products have phenotypes that can be measured readily or provide a growth advantage. For example, the screening of random DNA fusions to lacZ (encoding fl-galatosidase) identified bacterial genes whose expression changes in response to such varied stimuli as nutrient deprivation, heat stress, DNA damage, and varied carbon source.

1 mM ZnC12, and 1 mM iodoacetamide in 13 × 100-mm glass tubes. Include a blank without cells. 1% sodium dodecyl sulfate and 50 /~1 chloroform, vortex, and incubate at 37 ° for 5 min to permeabilize cells. Place tubes on ice for 5 min to cool. 0) to each individual tube, agitate, and place in 37 ° water bath. Note time. 0, 1 M KH2PO4, and place the tube in an ice-water bath to stop the reaction. Note time. Measure OD550 and OD420for each sample, using an assay mixture without cells as a blank. 75 x OD55o)] x 1000 Units activity = time (min) × OD6oo X volume cells (ml) Acknowledgments We thank J.

6 Like lactose MacConkey agar, this rich medium takes advantage of the acidic by-products formed by cells that can ferment lactose, although the way in which this works is a little more convoluted. All cells, which can grow on lactose tetrazolium agar, reduce the tetrazolium (2,3,5triphenyl-2-N-tetrazolium chloride) to form a red, insoluble formizan dye. 24 j. p. Horwitz, J. Chua, R. J. Curby, A. J. Tomson, M. A. DaRooge, B. E. Fisher, J. Mauricio, and I. Klundt, J. Med. Chem. 7, 574 (1964). 32 GENEFUSIONS [21 The acid by-products secreted by Lac ÷ colonies inhibit the reduction, preventing formation of the formizan.

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Applications of Chimeric Genes and Hybrid Proteins by Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.)

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