Understanding Recombinant DNA Technology: Transformation, Plasmids, and GFP Applications
This chapter provides an overview of recombinant DNA technology focusing on transformation, transfection, conjugation, and transduction methods to produce plasmids, specifically using the pGLO plasmid. It details the steps for growing bacterial colonies, DNA purification through miniprep and chromatography, and understanding key plasmid features, including antibiotic resistance, promoters, and terminators. Additionally, it explores the significance of green fluorescent protein (GFP) as a visual marker in studying biological processes and genetic modifications.
Understanding Recombinant DNA Technology: Transformation, Plasmids, and GFP Applications
E N D
Presentation Transcript
Recombinant DNA technology • Transformation • Transfection • Conjugation • Transduction
To get more plasmids • 1. Transformation – S3 and pGLO • Activity 5.1 and 5.3 • 2. Grow bacteria colonies in broth • 3. Purify DNA = miniprep • 4. Quantitate DNA purify GFP using column chromatography
DNA RNA Protein Trait
Experiments TRANSFORMATION Activity 5.1 and 5.2 PURIFY GFP BY CHROMATOGRAPHY Activity 7.3
What are plasmids? • Extrachromosomal pieces of DNA • Separate from the chromosomal DNA of bacteria • Name of each plasmid begins with p = plasmid • We will use a plasmid called pGLO
Different parts of a plasmid • Origin of replication • To copy themselves, recognition sites for DNA polymerases • Restriction enzyme sites (multiple cloning sites) • Sites to insert foreign DNA • Need to match plasmid with piece of DNA to clone
Different parts of a plasmid (continued) • Antibiotic resistance gene • Are passed between bacteria • Generate bacteria that are resistant to antibiotics • b-lactamses = bla • Breakdown antibiotics with b-lactam ring • Penicillin • Ampicillin • AmpR
Different parts of a plasmid (continued) • Promoter • Terminator
Selection • Separate the bacteria containing the plasmids from those that do not • After transformation • Some bacteria will have plasmid and some will not • Grow bacteria with plasmid on LB + Amp • Will bacteria grow? • Grow bacteria without plasmid on LB + Amp • Will bacteria grow?
Types of plasmids • Look at figure 5.3 • Plasmids can be used for protein production or genetic modifications • Expression plasmid • Plasmid used to express recombinant proteins • pGLO plasmid • Express GFP = green fluorescent protein • Cloning plasmid • Plasmid used to house genes
What is GFP? • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms
