University:
Boston University
Course:
CAS BI 203 | Cell Biology
Academic year:

2023
Views:

102

Pages:

18
Author:

alzheimer24ozql

Workshop Time Line • Introduction • Transform bacteria with pGLO plasmid Central Framework of Molecular Biology DNA RNA Protein Trait Links to Real-world • 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 Using GFP as a biological tracer pGLO Bacterial Transformation Kit Transformation Procedure Overview Day 1 What is Transformation? • Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance What is a plasmid? • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or be modified to express proteins of interest Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid Gene Expression • Beta Lactamase – Ampicillin resistance • Green Fluorescent Protein (GFP) – Aequorea victoria jellyfish gene • araC regulator protein – Regulates GFP transcription Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid Transcriptional Regulation 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 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 on ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates