Biorad pGlo-Biotechnology Explorer Program 11/20/2018 Helwig Serious About Science Education

Bacterial Transformation Molecular Biology Lab 6 Bacterial Transformation p GLO 11/20/2018 Helwig

Links to real world study of biological processes (ex. biosynthesis of proteins) localization of gene expression cell movement cell fate during development formation of different organs screen-able marker to identify transgenic organisms 11/20/2018 Helwig

Green Fluorescent Protein Aequorea victoria 11/20/2018 Helwig

What makes jelly fish glow? Calcium: the body's conductor Why can photo proteins, the flash-bulb proteins that jellies such as Aequorea Victoria turn on to give the outer edge of the creature's bell-shaped body a bluish or greenish glow? The answer centers on calcium. The jelly's light switch turns on in the presence of calcium ions, and the calcium ion is one of nature's favorite intercellular messengers, Whenever you tense a muscle or waggle a finger; whenever your heart pounds out a beat, pulsing blood through a tangle of veins and arteries, calcium has prompted the muscle to act, contract or twitch. 11/20/2018 Helwig

Alba the GFP Bunny Alba the GFP Bunny 11/20/2018 Helwig

What is transformation? Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance 11/20/2018 Helwig

What is a plasmid? A circular piece of autonomously replicating DNA ori bla Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest 11/20/2018 Helwig

pGlo plasmid Beta Lactamase Green Fluorescent Protein Ampicillin resistance The beta lactamase protein is produced and secreted by bacteria that contain the plasmid. Inactivates ampicillin. Green Fluorescent Protein Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription pGLO ori bla GFP araC 11/20/2018 Helwig

Bacterial Transformation GFP Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall 11/20/2018 Helwig

Bacterial DNA Genomic Bacterial cell Plasmid DNA Genomic DNA 11/20/2018 Genomic DNA Helwig

Transcription regulation HELWIG Transcription regulation Lactose operon Arabinose operon pGLO plasmid pGLO ori bla GFP araC 11/20/2018 Helwig

ARABINOSE SOURCE OF ENERGY AND CARBON FOR BACTERIA. FOUND IN PLANTS AND IS NORMALLY USED AS A SOURCE FOOD FOR BACTERIA. THE BACTERIAL GENES THAT MAKE DIGESTIVE ENZYMES TO BREAK DOWN ARABINOSE FOR FOOD ARE NOT EXPRESSED WHEN ARABINOSE IS ABSENT. WHEN ARABINOSE IS PRESENT GENES TURN ON. WHEN ABSENT GENES TURN OFF. ARABINOSE INITIATES TRANSCRIPTION OF THESE GENES BY PROMOTING THE BINDING OF RNA POLYMERASE. 11/20/2018 Helwig

ARABINOSE OPERON 11/20/2018 Helwig

Expression of Green Fluorescent Protein 11/20/2018 Helwig

Ara Operon and ara GFP Operon RNA Polymerase araC ara GFP Operon GFP Gene ara Operon araC B A D Effector (Arabinose) Effector (Arabinose) araC B A D RNA Polymerase araC B A D 11/20/2018 Helwig

lac Operon and ara operon Z Y A LacI Effector (Lactose) lac Operon B A D araC RNA Polymerase Effector (Arabinose) ara Operon RNA Polymerase 11/20/2018 Helwig

Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent cells uptake DNA after heat shock 11/20/2018 Helwig

Transformation procedure Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42oC and place on ice Incubate with nutrient broth Streak plates 11/20/2018 Helwig

Reasons for performing each transformation step Ca++ O CH2 P Base OH Sugar Transformation solution = CaCl2 pH = 6.1 Positive charge of Ca+2 ions neutralizes negative charge of phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to enter the cells. 11/20/2018 Helwig

Why perform each transformation step? Incubation on ice slows fluid cell membranes Heat-shock increases the permeability of cell membrane to DNA. Increase the bacterial uptake of foreign DNA. Nutrient broth incubation allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance) 11/20/2018 Helwig

What is nutrient broth or LB broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression carbohydrates amino acids nucleotides salts vitamins 11/20/2018 Helwig

RECOVERY Recovery is the 10 minute incubation period after you added the LB which allows the cells to grow and express the ampicillin resistant beta galactamase so the newly transformed cells survive on ampicllin plates. 11/20/2018 Helwig

Which colonies will glow? Grow? Glow? Which colonies will glow? Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp LB/Amp/Ara LB 11/20/2018 Helwig

How do I measure 250 micro liters using the pipette? Volume Measurement 11/20/2018 Helwig

Central Framework of Molecular Biology DNA RNA Protein Trait 11/20/2018 Helwig