BioRad pGLO: Transform bacteria with a Jellyfish gene to make them glow Module based on a kit from Bio-Rad Laboratories, Inc. Thank you to : Adapted by Dan Murray from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA.

Source of “glowing gene” for this experiment Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other Critters

Outline Overview Bacteria and Plasmids Transformation The pGLO Plasmid Experimental Procedures Extension Activities

Overview

What is Bacterial Transformation? Taking up of DNA from the environment by bacterial cells

Bacterial Transformation Lab Bacterial Cells and plasmid DNA are mixed Cells take up plasmid Cell/DNA mix is plated on nutrient agar with antibiotic Only cells which obtained plasmid DNA will grow… and glow

Bacteria and Plasmids

What is a plasmid? Small circular DNA molecule Replicates autonomously Originally evolved in bacteria May contain antibiotic resistance gene or be modified to contain other genes bla is an ampicillin resistance gene ori bla

Bacterial Cells and DNA Chromosomal Bacterial cell Chromosomal DNA Plasmid DNA

Growth of Bacteria on Plates Agarose in Petri dish = plate Incubate at 37C If few cells grow If many cells grow colonies lawn

Transformation

Bacterial Transformation The uptake of DNA Bacterial Cell Chromosomal DNA Plasmids

Methods of transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat Shock Chemically-competent cells uptake DNA after heat shock

The pGLO Plasmid

pGLO Plasmid bla gene GFP gene araC gene ori beta-lactamase enzyme Ampicillin resistance GFP gene Green Fluorescent Protein Aequorea victoria jellyfish araC gene On/off switch that reacts to arabinose ori Allows plasmid replication pGLO ori bla GFP araC

pGLO Plasmid: Most Important Components bla gene Bacteria with this gene grow in the presence of ampicillin GFP gene Bacteria with this gene glow under near UV light pGLO GFP bla

Experimental Procedures

Transformation Procedures +CaCl2 +CaCl2

Transformation Procedures

Reasons for Each Transformation Step Ca++ O CH2 P Base OH Sugar CaCl2 treatment Positive charge of Ca2+ ions neutralizes: negative charge of DNA phosphates negative charge of membrane phospholipids

Reasons for Each Transformation Step Incubation on ice slows fluid cell membranes Heat-shock increases permeability of cell membrane Nutrient broth incubation allows beta lactamase expression video

Purpose of each plate -pGLO/LB = Control -pGLO/LB/amp = tests the effect of ampicillin +pGLO/LB/amp = shows that ampicillin resistance has been acquired +pGLO/LB/amp/ara = shows that both traits have been acquired

Transformation Results Only cells getting pGLO plasmid grow and glow CONTROL All cells grow since there is no antibiotic on the plate Without pGLO plasmid, nothing can grow All cells grow since there is no antibiotic on the plate

Extension Activities

Extension Activity I: Transcriptional Regulation Arabinose controls expression of GFP gene: Transfer Bacteria Glowing Bacteria from Transformation Plate with Arabinose Plate without Arabinose Incubate overnight @ 37C

Extension Activity I: Transcriptional Regulation arabinose = no glow +arabinose = glow After overnight incubation Plate with Arabinose Plate without Arabinose

Transcriptional Regulation of GFP by Arabinose araC GFP Gene araC repressor blocks transcription Arabinose araC GFP Gene Arabinose binds repressor, changing its conformation RNA Polymerase araC GFP Gene Altered repressor leaves DNA, RNA polymerase can perform transcription

Extension Activity II: Tweaking the Transformation Protocol Test effect of various components of the transformation protocol: plate ampicillin concentration plate arabinose concentration amount of plasmid DNA used in the experiment amount of cells used in the experiment length of time cells/DNA mix is kept at 42C during the experiment Compare results with number of colonies obtained during the normal protocol

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