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This Little Light of Mine: Transform bacteria with a Jellyfish gene to make them glow

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

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This Little Light of Mine: Transform bacteria with a Jellyfish gene to make them glow

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  1. Module based on a kit from Bio-Rad Laboratories, Inc. Adapted by Dan Murray from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA. This Little Light of Mine:Transform bacteria with a Jellyfish gene to make them glow

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

  3. Jellyfish Gene put into Other Critters

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

  5. Overview

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

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

  8. Bacteria and Plasmids

  9. What is a plasmid? ori bla • 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

  10. Bacterial Cells and DNA Plasmid DNA Chromosomal Chromosomal DNA Bacterial cell

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

  12. Transformation

  13. Chromosomal DNA Bacterial Cell Bacterial Transformation Plasmids The uptake of DNA

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

  15. The pGLO Plasmid

  16. pGLO Plasmid araC ori pGLO GFP bla • bla gene • beta-lactamase enzyme • Ampicillin resistance • GFP gene • Green Fluorescent Protein • Aequorea victoria jellyfish • araC gene • Regulates GFP transcription • ori • Allows plasmid replication

  17. 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

  18. pGlo Plasmid Additional Features

  19. 1. Arabinose sugar binds regulator. 2. Regulator plus arabinose complex recruits RNA Polymerase to bind promotor. 3. Gene is transcribed into mRNA and “read.”

  20. Experimental Procedures

  21. Transformation Procedures +CaCl2 +CaCl2

  22. Transformation Procedures

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

  24. Reasons for Each Transformation Step • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Nutrient broth incubationallows beta lactamase expression

  25. Transformation Results Lb/amp/ara Only cells getting pGLO plasmid grow and glow 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 White – no glow

  26. Extension Activities

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

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

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

  30. Compare results with number of colonies obtained during the normal protocol 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

  31. Biotechnology Explorer Program Serious About Science Education

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