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Chapter 5 background

Chapter 5 background. 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

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Chapter 5 background

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  1. Chapter 5 background

  2. Recombinant DNA technology • Transformation • Transfection • Conjugation • Transduction

  3. 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

  4. DNA RNA Protein Trait

  5. Experiments TRANSFORMATION Activity 5.1 and 5.2 PURIFY GFP BY CHROMATOGRAPHY Activity 7.3

  6. 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

  7. pGLO plasmid

  8. 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

  9. 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

  10. Different parts of a plasmid (continued) • Promoter • Terminator

  11. 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?

  12. 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

  13. 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

  14. GFP

  15. Using GFP as a biological tracer

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