1. This Little Light of Mine: 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.

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

8. Bacteria and Plasmids

9. 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

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

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

12. Transformation

13. Bacterial Transformation Plasmids Chromosomal DNA Bacterial Cell 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 ori bla GFP araC pGLO Plasmid 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 bla GFP 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

18. 18 pGlo Plasmid Additional Features

19. 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. 20

21. Experimental Procedures

22. Transformation Procedures +CaCl 2

23. Transformation Procedures

24. Reasons for Each Transformation Step  CaCl 2 treatment Positive charge of Ca 2+ ions neutralizes: negative charge of DNA phosphates negative charge of membrane phospholipids Ca ++ O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O Ca ++

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

26. Transformation Results 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 Lb/amp/ara White – no glow

27. Extension Activities

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

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

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

31. 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

32. Biotechnology Explorer Program Serious About Science Education