pglo lab presentation - ap biology with mrs. haas...history • 1928-griffith showed harmless...
TRANSCRIPT
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!