221 Transformation of E. coli with pGALâ¢ (blue colony) - EDVOTEK

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EDVO-Kit # 221Transformation of E. coli with pGAL (blue colony)Bacterial TransformationBackground InformationPvu IIRru IPvu IMst IBgl IoriXmn Iamp rpGAL6751 bpEco RIBam HIEco RIHind IIIerally range from 1 x 10 4 to 1 x 10 7 cells per microgram of DNA. Thereare special procedures which can produce cells having transformationefficiencies approaching 10 10 . However, transformation is never 100%efficient. Approximately 1 in every 10,000 cells successfully incorporatesplasmid DNA in preparations having average competency. However,there is such a large number of cells in a sample (typically 1 x 10 9 ) thatonly a small fraction needs to be transformed to obtain colonies on aplate. The same volume of recovered cells plated on selective (containsantibiotic) and nonselective agar medium will yield vastly different numbersof cells. The nonselective medium will have many more growingcells that form a bcterial lawn.ß-GalactosidaseFigure 2 :DNA map of pGALNot all restriction enzymes are shown.Many different plasmids serve as useful tools inmolecular biology. One example is the pGALplasmid, present in multiple copies in specifiedhost E. coli host cells. It contains 6751 basepairs and has been cleverly modified by geneticengineering. In the cell, it does not integrateinto the bacterial chromosome, but replicatesautonomously. The pGAL plasmid contains theE. coli gene which codes for β-galactosidase. Inthe presence of artificial galactosides such as5-Bromo-4 Chloro 3-indolyl-β-D-galactoside (X-Gal), pGAL colonies appear blue when X-Gal iscleaved by β-galactosidase and forms a coloredproduct.This experiment has been designed to utilizeEDVOTEK Transformation LyphoCells. It alsocontains the proprietary plasmid, pGAL (BlueColony), which was engineered by EDVOTEK.Plasmid pGAL carries the complete gene forβ‐galactosidase. Since the host E. coli does notcontain a β-galactosidase gene, only cells transformedby the pGAL plasmid will produce thefunctional β‐galactosidase enzyme. Cells thatexpress β-galactosidase will cleave X-Gal and thepGAL transformed colonies will be blue.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose withoutthe written consent of EDVOTEK, Inc. Copyright © 1989, 90, 93, 94, 97, 98, 2000, 2005, EDVOTEK, Inc., all rightsreservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with pGAL (blue colony)EDVO-Kit # 221Bacterial TransformationIn addition to the expression and cleavage of X-Gal by β-galactosidase,transformation by pGAL is also demonstrated by resistance to ampicillin.E. coli host cells used in this experiment are not naturally resistantto ampicillin. The plasmid pGAL contains the gene which encodes forβ-lactamase that inactivates ampicillin. E. coli cells transformed by pGALwill express the resistance gene product β-lactamase as an extracellularenzyme excreted from E. coli cells. Once outside the cell, the enzymediffuses into the surrounding medium and inactivates ampicillin.With time, small "satellite" colonies may appear around a large blue colony.Cells in the small "satellite" or "feeder" colonies are not resistantto ampicillin and have not been transformed with the pGAL plasmid.They are simply growing in a region of agar where β-lactamase has diffusedand inactivated the antibiotic ampicillin. The number of satellitecolonies increases if the concentration of ampicillin is low or the plateshave incubated for longer times.The ExperimentDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose withoutthe written consent of EDVOTEK, Inc. Copyright © 1989, 90, 93, 94, 97, 98, 2000, 2005, EDVOTEK, Inc., all rightsreservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
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221 Transformation of E. coli with pGALâ¢ (blue colony) - EDVOTEK