This document provides information about using a pGLO bacterial transformation kit for teaching purposes. It discusses how bacterial transformation and protein purification are powerful teaching tools that allow students to make real-world connections. The document then summarizes the key steps and components of the pGLO bacterial transformation procedure, including transforming bacteria with a plasmid containing the GFP gene, growing transformed colonies, and inducing GFP expression.

2. pGLO™ Transformation

3. WhyTeachBacterial Transformationand Protein Purification? Powerful teaching tool Laboratory extensions Real-world connections Link to careers and industry Standards based

6. pGLO™ Bacterial Transformation Kit Bio-Rad pGLO Kit Advantages Standards-based Comprehensive curricula for inquiry-based investigations Compatible with 50 minute class periods Serves entire class of 32 students (up to 4 students per group) Cost-effective Success in student’s hands Safe Striking results!

9. En procariontes El promotor es la secuencia que señala el comienzo de la transcripción del ADN a ARN, y es por ello el lugar de enlace de la ARN polimerasa. El promotor se encuentra en una de las dos hebras del ADN, y la orientación y posición del promotor dictará cuál de las dos hélices del ADN servirá como molde para sintetizar el ARN. Muchos promotores necesitan de proteínas activadoras antes de que puedan unirse eficazmente a su respectiva polimerasa. Generalmente, el enlace de la ARN polimerasa con el promotor es lo que facilita a que la hélice de ADN se abra para permitir la subsecuente transcripción a ARN.

10. 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

12. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

13. pGLO Bacterial Transformation Kit

14. Transformation

15. Transformation 2 Day 1 Day 2

17. can’t glow

18. competent - able to be transformedBacterial chromosome

21. AmpR Ara GFP Transformation E. coli cells Plasmid Jellyfish DNA

22. AmpR GROW ON AN AGAR PLATE GFP Ara … that can GLOW! Transformation Recombinant Bacteria…

23. makes all transformed bacteria resistant to ampicillin controls GFP gene expression only turned on in the presence of arabinose Transformation AmpR pGLO plasmid GFP Ara promoter

24. What is Transformation? GFP Uptake of foreign DNA, often a circular plasmid Beta-lactamase Ampicillin Resistance

25. 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

26. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

27. The Many Faces of Plasmids Graphic representation Scanning electron micrograph of supercoiled plasmid

29. Ampicillin resistance

30. Green Fluorescent Protein (GFP)

31. Aequorea victoria jellyfish gene

32. araC regulator protein

34. Transcriptional Regulation Lactose operon Arabinose operon pGLO plasmid

35. ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D Z Y A LacI RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation

36. ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation

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

38. 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

39. Reasons for Performing Each Transformation Step? Ca++ O Ca++ P O O Base O O CH2 Sugar Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O O P Base O O CH2 Sugar OH

40. Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance)

41. What is Nutrient Broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins

42. LB/Amp LB/Amp/Ara LB Grow?Glow? Follow protocol On which plates will colonies grow? Which colonies will glow?

43. LaboratoryQuick Guide