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Recombinant DNA technologyTransformation
Transfection
Conjugation
Transduction
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To get more plasmids1. 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
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DNA RNA Protein Trait
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ExperimentsTRANSFORMATION
Activity 5.1 and 5.2
PURIFY GFP BY CHROMATOGRAPHY
Activity 7.3
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What are plasmids?Extrachromosomal pieces of DNASeparate from the chromosomal DNA of
bacteriaName of each plasmid begins with p =
plasmidWe will use a plasmid called pGLO
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pGLO plasmid
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Different parts of a plasmidOrigin of replication
To copy themselves, recognition sites for DNA polymerases
Restriction enzyme sites (multiple cloning sites)Sites to insert foreign DNANeed to match plasmid with piece of DNA to
clone
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Different parts of a plasmid (continued)
Antibiotic resistance geneAre passed between bacteriaGenerate bacteria that are resistant to
antibiotics-lactamses = blaBreakdown antibiotics with -lactam ring
Penicillin Ampicillin
AmpR
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Different parts of a plasmid (continued)
Promoter
Terminator
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SelectionSeparate the bacteria containing the plasmids
from those that do not
After transformationSome 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?
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Types of plasmidsLook at figure 5.3
Plasmids can be used for protein production or genetic modifications
Expression plasmidPlasmid used to express recombinant proteinspGLO plasmid
Express GFP = green fluorescent protein
Cloning plasmidPlasmid used to house genes
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What is GFP?GFP is a visual markerStudy of biological processes (example:
synthesis of proteins)Localization and regulation of gene
expressionCell movementCell fate during developmentFormation of different organsScreenable marker to identify transgenic
organisms
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GFP
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Using GFP as a biological tracer