chapter 5 background

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