chapter 5 background
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
Recombinant DNA technologyTransformation
Transfection
Conjugation
Transduction
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
DNA RNA Protein Trait
ExperimentsTRANSFORMATION
Activity 5.1 and 5.2
PURIFY GFP BY CHROMATOGRAPHY
Activity 7.3
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
pGLO plasmid
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
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
Different parts of a plasmid (continued)
Promoter
Terminator
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?
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
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
GFP
Using GFP as a biological tracer