Bacterial Transformation

pGLO Lab

Bacterial Transformation

• uptake and incorporation of external DNA into a bacterial cell

• may occur naturally or artificially

History

• 1928-Griffith showed harmless Strep. pneumoniae could become virulent after contact with heat-killed virulent strains

• 1944-Avery, MacLeod, McCarty showed DNA was “culprit”

Structure of Bacteria

Notice both nucleoid DNA

and plasmid DNA

Background Info.

• Genes can be moved into a cell by plasmid

• plasmid = extra loop of DNA, usually carries beneficial traits; not always present; source of genetic diversity

• Remember: bacteria have one large, circular chromosome

The Many Faces of Plasmids

Scanning electron micrograph of supercoiled plasmid

Graphic representation

pGLO

• using plasmid containing gene for bioluminescence from jellyfish

• when expressed, created green florescent protein (GFP)

• plasmid also contains gene for ampicillin resistance (antibiotic)

• relies on operon system

• operon system is turned on by arabinose sugar

Transcriptional Regulation

•Lactose operon

•Arabinose operon

•pGLO plasmid

Operon

• group of genes controlled by one promoter

• ex: lac operon

Review!!

Why keep this operon off when lactose is absent?

Procedure

• Prepare cells (make them competent) for plasmid pick up

• Add plasmid

• Incubate cells for proliferation

Reasons for Performing Each Transformation Step?

1.Transformation solution = CaCI2

Positive charge of Ca++ ions shields negative charge of DNA phosphates

Ca++

Ca++

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Why Perform Each Transformation Step?

2. Incubate on ice slows fluid cell

membrane

3. Heat-shock Increases permeability of

membranes

4. Nutrient broth incubation

Allows beta-lactamase expression

Beta-lactamase (ampicillin resistance)

Cell wall

GFP

Bacterial Transformation

Beta lactamase (ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

Incubation

• Transformed bacteria (or not) will be grown on various plates to determine the environmental impact on GRP and AMP resistance expression

Before Lab...• Please read the entire lab packet.

• Recommended: Answer packet intro questions for better clarification.

• Have at least objective and hypothesis done.

• Find a lab station, be familiar with what and where the materials are!

• Ask questions as needed!