221 Transformation of E. coli with pGAL™ (blue colony) - EDVOTEK

Transformation of E. coli with pGAL (blue colony)EDVO-Kit # 221Bacterial TransformationBacterial transformation is of central importance in molecular biology.It allows for the introduction of genetically engineered or naturally occurringplasmids in bacterial cells. This makes possible the propagation,genetic expression and isolation of DNA plasmids.The transformation process involves the uptake of exogenous DNA bycells which results in a newly acquired genetic trait that is stable andheritable. Bacterial cells must be in a particular physiological statebefore they can be transformed. This state is referred to as competency.Competency can occur naturally in certain species of Haemophilus andBacillus when the levels of nutrients and oxygen are low. CompetentHaemophilus expresses a membrane associated transport complex whichbinds and transfers certain DNA molecules from the medium into thecell where they are incorporated and their genes are expressed. In nature,the source of external DNA is from other cells.Most of the current transformation experiments involve E. coli. This organismdoes not enter a stage of competency unless artificially induced.Treatment to achieve competency involves the use of chloride salts, suchas calcium chloride, and sudden hot and cold temperature changes. Themetal ions and temperature changes affect the structure and permeabilityof the cell wall and membrane so that DNA molecules can beabsorbed by the bacteria. The mechanism of DNA transport in the cellstill is not fully understood. Competent E. coli cells are fragile and mustbe treated carefully.Background InformationNumber oftransformantsµg of DNA100transformants0.01 µgXSpecific example:Xfinal vol atrecovery (ml)vol plated (ml)1 ml0.1 mlFigure 1:Bacterial Transformation Efficiency Calculation==Number oftransformantsper µg100,000(1 x 10 5 )transformantsper µgThe transformation efficiency is defined by the number of transformantsobtained per microgram of DNA. For example, 10 nanograms of DNAwere used for a transformation and the cells were allowed to recover ina final volume of 1 ml. One tenth of this volume was plated and produced100 colonies on a selective agarmedium. Therefore, 1000 transformantsare present per ml. Keeping inmind that each colony grew from onetransformed cell, the efficiency wouldbe 1000/0.01ug = 1 x 10 5 . Transformationefficiencies of 10 5 to 10 6 are morethan sufficient for most subcloningexperiments. When the cloning ofsingle copy genes from genomic DNAis done, the required efficiencies are10 7 to 10 8 .The determination for transformationefficiency in this case is outlined in Figure1. Transformation efficiencies gen-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