p GLO GFP Central Framework of Molecular Biology
p. GLO™ & GFP
Central Framework of Molecular Biology The Central Dogma as proposed by Francis Crick DNA RNA Protein Trait
Links of this Lab to Realworld • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms
Transformation Procedure Day 1 Day 2
Bacterial DNA Images Bacterial cell Plasmid DNA Genomic DNA
• What is Transformation? Uptake of foreign DNA, often a circular plasmid • The plasmid that we want the bacteria to uptake in this lab is called p. GLO. • There are two important genes found on this operon: a. One called AMP that codes for the enzyme beta-lactamase (the enzyme that allows the bacteria to be resistant to amoxicillin – an antibiotic. b. One called GFP that codes for the green flourescent protein (allows the bacteria to glow under certain conditions. ) GFP Beta-lactamase Ampicillin Resistance
Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) p. GLO plasmids
Volume Measurement
What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium (Food) that contains nutrients for bacterial growth and gene expression – Carbohydrates – Amino acids – Nucleotides – Salts – Vitamins • The petri dishes are made using LB broth. For this lab you will be using three different dishes: a. Some of the dishes will just have the LB b. Some will have arabinose added to the broth, and c. Some dishes will have both arabinose and ampicillin added.
Transcriptional Regulation: Modification of the normal Arabinose Operon • Lactose operon • Arabinose operon • p. GLO plasmid
Transcriptional Regulation: The lac and ara (Arabinose Operon) are both inducible. The effector is another name for the INDUCER. ara Operon lac Operon Lac. I Z Y A ara. C Y A B A D RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) Lac. I B ara. C B A D
Gene Regulation: How the ara Operon has been modified. The B-A-D structural genes have been enzymatically removed from the operon and have been replaced by the GFP Gene. ara GFP Operon ara. C B A D ara. C Effector (Arabinose) ara. C B A D ara. C RNA Polymerase ara. C B A D GFP Gene RNA Polymerase ara. C GFP Gene
Methods of Transformation • Electroporation – Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat-Shock – Chemically-competent cells uptake DNA after heat shock
Transformation Procedure • Suspend bacterial colonies in Transformation solution • Add p. GLO plasmid DNA • Place tubes in ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates
Reasons for Performing Each Transformation Step? Ca++ O O P O O CH 2 Base O Sugar 1. Transformation solution = Ca. CI 2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O O CH 2 Base O Sugar OH
Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes to allow for plasmid uptake 4. Nutrient broth incubation Allows beta-lactamase expression (transcription/transl. of the gene will occur) Beta-lactamase (ampicillin resistance)
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