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

More documents

Recommendations

Info

EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsBacterial TransformationBackground InformationSupercoiledRelaxedFigure 2: Supercoiled andcircular forms of plasmid DNAsare called "satellites", since they only appear around larger colonies oftransformed cells. Larger plating volumes and longer incubation timesincrease the number of satellite colonies.Plasmids naturally exist as supercoiled molecules. The twostrands of DNA in the supercoiled molecule wind around eachother to produce a condensed, entangled structure when comparedto relaxed (non-supercoiled) DNA (Figure 2). CompetentE. coli cells are sensitive to the conformation of the DNA theywill accept. Supercoiled DNA gives the highest transformationefficiencies.SupercoiledpFluoroGreenDNABacterialchromosomal DNAGreenfluorescentproteinFigure 3Overview of GFP and BFP ExpressionIn this experiment, the goal is to express fluorescent proteins (GFP andBFP) in transformed bacterial cells. To begin this process, there must bea means of "turning on" the cloned GFP or BFP gene in the recombinantplasmid. In order to have an "off/on" switch for controlling expression,the gene is placed under the control of a DNA sequence known as a"promoter".A promoter is a sequence in DNA that typically occurs just in front("upstream") of the DNA coding sequence (the sequence that specifiesthe amino acid sequence for a protein). The chromosome of the hostbacterial strain used in this experiment has been genetically engineeredto contain the gene for RNA polymerase, which is under control of thelac promoter, and can be turned on (induced) by the presence of a smallmolecule called IPTG (isopropyl-beta-D-thiogalactopyranoside). IPTGbinds to and inactivates an inhibitor protein known as the lac repressor.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 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 TransformationThe sequence of events required to turn on expression of GFP/BFP is asfollows:1. Cells are grown in the presence of IPTG (to turn on the lac promoter)which binds to, and releases the lac repressor that sits on apromoter (lac) upstream of the RNA polymerase gene. The releaseof the inhibitor allows the RNA polymerase to be produced from theE. coli genome.2. The RNA polymerase, in turn, recognizes the promoter on theplasmid mRNA enabling production of large quantities of thefluorescent protein. In summary, a strong promoter, combined withan active RNA polymerase, allows for very high levels of GFP or BFPmRNA (and thus protein) expression in the transformed cells.Background InformationDuplication 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
Page 5 and 6: Transformation of E. coli with Gree
Page 7: Transformation of E. coli with Gree
Page 21: Transformation of E. coli with Gree
Page 24 and 25: 24EDVO-Kit # 222Transformation of E

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

Create successful ePaper yourself

Delete template?

Save as template?