pGLO™ Transformation<br />
WhyTeachBacterial Transformationand Protein Purification?<br />Powerful teaching tool<br />Laboratory extensions<br />Real...
pGLO™ Bacterial Transformation Kit<br />Bio-Rad pGLO Kit Advantages<br />Standards-based<br />Comprehensive curricula for ...
WorkshopTime Line<br /><ul><li>Introduction
Transform bacteria with pGLO plasmid</li></li></ul><li>DNA<br />RNA<br />Protein<br />Trait<br />Central Framework of Mole...
En  procariontes<br />El promotor es la secuencia que señala el comienzo de la transcripción del ADN a ARN, y es por ello ...
Links to   Real-world<br />GFP is a visual marker<br />Study of biological processes 		(example: synthesis of proteins)<br...
Using GFP as a biological tracer<br />http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html<br />With permission from ...
pGLO Bacterial Transformation Kit<br />
Transformation<br />
Transformation 2<br />Day 1<br />Day 2<br />
E. coli cells<br /><ul><li> sensitive to antibiotics
 can’t glow
competent - able to be transformed</li></ul>Bacterial chromosome<br />
AmpR<br />Ara<br />promoter<br />Transformation<br />Plasmid containing:<br /><ul><li> Ampicillin resistance gene (always ...
 Ara promoter - turned on in the presence of arabinose</li></li></ul><li>GFP gene<br />Transformation<br />Jellyfish DNA<b...
AmpR<br />Ara<br />GFP<br />Transformation<br />E. coli cells<br />Plasmid<br />Jellyfish DNA<br />
AmpR<br />GROW ON AN AGAR PLATE<br />GFP<br />Ara<br />… that can GLOW!<br />Transformation<br />Recombinant Bacteria…<br />
makes all transformed bacteria resistant to ampicillin<br />controls GFP gene expression<br />only turned on in the presen...
What is Transformation?<br />GFP<br />Uptake of foreign DNA, often a circular plasmid<br />Beta-lactamase<br />Ampicillin ...
What is a plasmid?<br />A circular piece of autonomously replicating DNA<br />Originally evolved by bacteria<br />May expr...
Bacterial DNA<br />Bacterial cell<br />Plasmid DNA<br />Genomic DNA<br />
The Many Faces of Plasmids<br />Graphic representation<br />Scanning electron micrograph of supercoiled plasmid<br />
GeneExpression<br /><ul><li>Beta Lactamase
Ampicillin resistance
Green Fluorescent Protein (GFP)
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription</li></li></ul><li>Bacterial Transformation<br />Cell wall<br />GFP<br />Bacterial <br />chromo...
Transcriptional Regulation<br />Lactose operon<br />Arabinose operon<br />pGLO plasmid<br />
ara Operon<br />lac Operon<br />araC<br />B<br />A<br />D<br />LacI<br />Z<br />Y<br />A<br />Effector (Arabinose)<br />Ef...
ara GFP Operon<br />ara Operon<br />araC<br />GFP Gene<br />araC<br />B<br />A<br />D<br />Effector(Arabinose)<br />Effect...
Methods of Transformation<br />Electroporation<br />Electrical shock makes cell membranes permeable to DNA<br />Calcium Ch...
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Práctica de Transformación

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Práctica de Transformación

  1. 1.
  2. 2. pGLO™ Transformation<br />
  3. 3. WhyTeachBacterial Transformationand Protein Purification?<br />Powerful teaching tool<br />Laboratory extensions<br />Real-world connections<br />Link to careers and industry<br />Standards based<br />
  4. 4.
  5. 5.
  6. 6. pGLO™ Bacterial Transformation Kit<br />Bio-Rad pGLO Kit Advantages<br />Standards-based<br />Comprehensive curricula for inquiry-based investigations<br />Compatible with 50 minute class periods<br />Serves entire class of 32 students (up to 4 students per group)<br />Cost-effective<br />Success in student’s hands<br />Safe<br />Striking results!<br />
  7. 7. WorkshopTime Line<br /><ul><li>Introduction
  8. 8. Transform bacteria with pGLO plasmid</li></li></ul><li>DNA<br />RNA<br />Protein<br />Trait<br />Central Framework of Molecular Biology<br />
  9. 9. En procariontes<br />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.<br />Muchos promotores necesitan de proteínas activadoras antes de que puedan unirse eficazmente a su respectiva polimerasa.<br />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.<br />
  10. 10. Links to Real-world<br />GFP is a visual marker<br />Study of biological processes (example: synthesis of proteins)<br />Localization and regulation of gene expression<br />Cell movement<br />Cell fate during development<br />Formation of different organs<br />Screenable marker to identify transgenic organisms<br />
  11. 11.
  12. 12. Using GFP as a biological tracer<br />http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html<br />With permission from Marc Zimmer<br />
  13. 13. pGLO Bacterial Transformation Kit<br />
  14. 14. Transformation<br />
  15. 15. Transformation 2<br />Day 1<br />Day 2<br />
  16. 16. E. coli cells<br /><ul><li> sensitive to antibiotics
  17. 17. can’t glow
  18. 18. competent - able to be transformed</li></ul>Bacterial chromosome<br />
  19. 19. AmpR<br />Ara<br />promoter<br />Transformation<br />Plasmid containing:<br /><ul><li> Ampicillin resistance gene (always expressed)
  20. 20. Ara promoter - turned on in the presence of arabinose</li></li></ul><li>GFP gene<br />Transformation<br />Jellyfish DNA<br />GFP = Green Fluorescent Protein<br />glows under UV light<br />
  21. 21. AmpR<br />Ara<br />GFP<br />Transformation<br />E. coli cells<br />Plasmid<br />Jellyfish DNA<br />
  22. 22. AmpR<br />GROW ON AN AGAR PLATE<br />GFP<br />Ara<br />… that can GLOW!<br />Transformation<br />Recombinant Bacteria…<br />
  23. 23. makes all transformed bacteria resistant to ampicillin<br />controls GFP gene expression<br />only turned on in the presence of arabinose <br />Transformation<br />AmpR<br />pGLO plasmid<br />GFP<br />Ara<br />promoter<br />
  24. 24. What is Transformation?<br />GFP<br />Uptake of foreign DNA, often a circular plasmid<br />Beta-lactamase<br />Ampicillin <br />Resistance<br />
  25. 25. What is a plasmid?<br />A circular piece of autonomously replicating DNA<br />Originally evolved by bacteria<br />May express antibiotic resistance gene <br /> or be modified to express proteins of interest<br />
  26. 26. Bacterial DNA<br />Bacterial cell<br />Plasmid DNA<br />Genomic DNA<br />
  27. 27. The Many Faces of Plasmids<br />Graphic representation<br />Scanning electron micrograph of supercoiled plasmid<br />
  28. 28. GeneExpression<br /><ul><li>Beta Lactamase
  29. 29. Ampicillin resistance
  30. 30. Green Fluorescent Protein (GFP)
  31. 31. Aequorea victoria jellyfish gene
  32. 32. araC regulator protein
  33. 33. Regulates GFP transcription</li></li></ul><li>Bacterial Transformation<br />Cell wall<br />GFP<br />Bacterial <br />chromosomal DNA<br />Beta lactamase<br />(ampicillin resistance)<br />pGLO plasmids<br />
  34. 34. Transcriptional Regulation<br />Lactose operon<br />Arabinose operon<br />pGLO plasmid<br />
  35. 35. ara Operon<br />lac Operon<br />araC<br />B<br />A<br />D<br />LacI<br />Z<br />Y<br />A<br />Effector (Arabinose)<br />Effector(Lactose)<br />araC<br />B<br />A<br />D<br />Z<br />Y<br />A<br />LacI<br />RNA Polymerase<br />RNA Polymerase<br />B<br />A<br />D<br />araC<br />Z<br />Y<br />A<br />Transcriptional Regulation<br />
  36. 36. ara GFP Operon<br />ara Operon<br />araC<br />GFP Gene<br />araC<br />B<br />A<br />D<br />Effector(Arabinose)<br />Effector (Arabinose)<br />araC<br />B<br />A<br />D<br />araC<br />GFP Gene<br />RNA Polymerase<br />RNA Polymerase<br />B<br />A<br />D<br />araC<br />araC<br />GFP Gene<br />Gene Regulation<br />
  37. 37. Methods of Transformation<br />Electroporation<br />Electrical shock makes cell membranes permeable to DNA<br />Calcium Chloride/Heat-Shock<br />Chemically-competent cells uptake DNA after heat shock<br />
  38. 38. Transformation Procedure<br />Suspend bacterial colonies in Transformation solution<br />Add pGLO plasmid DNA<br />Place tubes on ice<br />Heat-shock at 42°C and place on ice<br />Incubate with nutrient broth<br />Streak plates<br />
  39. 39. Reasons for Performing Each Transformation Step?<br />Ca++<br />O<br />Ca++<br />P<br />O<br />O<br />Base<br />O<br />O<br />CH2<br />Sugar<br />Transformation solution = CaCI2<br />Positive charge of Ca++ ions shields negative <br />charge of DNA phosphates<br />O<br />Ca++<br />O<br />O<br />P<br />Base<br />O<br />O<br />CH2<br />Sugar<br />OH<br />
  40. 40. Why Perform Each Transformation Step?<br />Cell wall<br />GFP<br />2. Incubate on ice<br /> slows fluid cell membrane<br />3. Heat-shock<br /> Increases permeability of membranes<br />4. Nutrient broth incubation<br /> Allows beta-lactamase <br /> expression<br />Beta-lactamase<br />(ampicillin resistance)<br />
  41. 41. What is Nutrient Broth? <br />Luria-Bertani (LB) broth<br />Medium that contains nutrients for bacterial growth and gene expression<br />Carbohydrates<br />Amino acids<br />Nucleotides<br />Salts<br />Vitamins<br />
  42. 42. LB/Amp<br />LB/Amp/Ara<br />LB<br />Grow?Glow?<br />Follow protocol<br />On which plates will colonies grow?<br />Which colonies will glow?<br />
  43. 43. LaboratoryQuick Guide<br />

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