Transformation of E. coli with Green and Blue ... - Frederiksen

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EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsBacterial TransformationBackground Informationcan be used to help students to appreciate genetically inherited diseasesand the effects of accumulated mutations on aging and the onset ofvarious diseases.Bacterial transformation and Overexpression ofTransformed GenesBacterial transformation is of central importance in molecular biology.Transformation is the process by which a bacterium takes up and expressesexogenous DNA, resulting in a newly acquired genetic trait thatis stable and heritable. This exogenous DNA can be recombinant DNAmolecules that have been constructed in vitro, as well as natural DNAmolecules. Transformation is also of historical importance since it led tothe discovery by Oswald Avery, in 1944, that DNA was the genetic material.In that historical experiment, Avery and colleagues purified DNAfrom a lethal strain of Streptococcus pneumoniae, removing all proteinfrom the DNA. This DNA was then transformed into a harmless strainof the same organism. Injection of the transformed, formerly harmless,strain into mice resulted in their death.For transformation to occur, bacterial cells must be in a particular physiologicalstate, referred to as competency, in which the bacterial cellwall is made permeable to macromolecules such as DNA. Competencycan occur naturally in certain species of Haemophilus and Bacillus whenthe levels of nutrients and oxygen are low. Competent Haemophiluscells express a membrane-associated transport complex that binds andtransfers certain DNA molecules from the medium into bacterial cellsthat are then integrated into the bacterial chromosome and expressed.In nature, the source of the external DNA is from other cells that havedied and their cell walls lysed to release their DNA into the surroundingmedium.Much current research in molecular biology involves the transformationof E. coli, an organism that does not naturally enter a state of competency.E. coli can artificially be made competent when treated withchloride salts of the metal cations calcium, magnesium and rubidium.In addition, abrupt transitioning between heat and cold can induce competency.It is believed that metal ions and temperature changes affectthe structure and permeability of the cell wall and membrane, allowingDNA molecules to pass through the bacterial cell wall. Due to theirunstable cell walls, competent E. coli cells are fragile and therefore mustbe treated carefully.The number of cells transformed per 1 microgram (µg) of DNA is knownas the transformation efficiency. In practice, much smaller amounts ofDuplication 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 without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222Bacterial TransformationNumber oftransformantsµg of DNA100transformants0.01 µgXSpecific example:XDNA are used (5 to 100 nanograms, ng) since excessive DNA (>100 ng)inhibits the transformation process. For example, when 10 nanograms(0.01 microgram) of DNA was usedto transform cells that were in a finalfinal vol atrecovery (ml)vol plated (ml)1 ml0.1 mlFigure 1:Bacterial Transformation Efficiency Calculation==Number oftransformantsper µg100,000(1 x 10 5 )transformantsper µgvolume of 1 ml, and if 0.1 ml (100 µl)of these cells were plated on mediumthat contained antibiotic, only thecells that acquired the foreign DNAcould grow. This procedure is calledselection. After incubation (in thisexample) 100 colonies were found onthe plate. Realizing that each colonyoriginally grew from one transformedcell, the transformation efficiency inthis example is 10 5 (outlined in Figure1). In research laboratories, transformationefficiencies generally rangefrom 1 x 10 7 to 1 x 10 8 cells per microgramof DNA. Special procedures canproduce cells having transformationefficiencies approaching 10 10 .The ExperimentTransformation is never 100% efficient. Approximately one in every10,000 cells (of average competency) successfully incorporates exogenousDNA. However, based on the large number of cells (typically 1x 10 9 ), only a small number of cells are transformed to obtain visiblecolonies on agar plates.This concept can be demonstrated by plating the same volume of recoveredcells on selective and nonselective agar medium. The nonselectivemedium will have many more growing cells since all the untransformedcells survive, in addition to the transformed cells. The nonselectivebacterial agar plates will be covered heavily with untransformed cells,forming a “lawn”, in contrast to individual colonies obtained on theselective agar plate.To ferry foreign genes into bacteria, plasmids are usually used. Plasmidsare self-replicating extrachromosomal, double-stranded circular DNAmolecules found in many strains of bacteria. Many plasmids containgenes that provide resistance to various antibiotics, including tetracycline,kanamycin, and ampicillin (amp). Ampicillin is a derivative ofpenicillin that inhibits bacterial growth by interfering with the synthesisof bacterial cell walls. The product of the ampicillin resistance gene isthe enzyme b-lactamase. This enzyme is secreted by transformed cellsinto the surrounding medium, where it destroys ampicillin. Due tothis extracellular secretion, cells that are not transformed are able toundergo limited growth in the zones surrounding transformed, antibiotic-resistantcells. Colonies consisting of these untransformed cellsDuplication 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 without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
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