Transcript ara Operon
pGLO™ & 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 pGLO. • 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) pGLO 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 • pGLO plasmid Transcriptional Regulation: The lac and ara (Arabinose Operon) are both inducible. The effector is another name for the INDUCER. ara Operon lac Operon LacI Z Y A ara C Z Y A araC Y A B A D RNA Polymerase RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) LacI B araC 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 The B-A-D structural genes have been enzymatically removed from the operon and have been replaced by the GFP Gene.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 Operon ara C B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D GFP Gene GFP Gene RNA Polymerase araC 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 pGLO 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++ Ca++ O O P O O CH2 Base O Sugar 1. Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O Base O CH2 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)