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Biotechnology

Biotechnology. Explorer Program. Serious About Science Education. Aequorea victoria. DNA. RNA. Protein. Trait. Central Framework of Molecular Biology. Transformation is a natural process that Bacterial have evolved in order to obtain DNA from their environment.

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Biotechnology

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  1. Biotechnology Explorer Program Serious About Science Education

  2. Aequorea victoria

  3. DNA RNA Protein Trait Central Framework of Molecular Biology

  4. Transformation is a natural process that Bacterial have evolved in order to obtain DNA from their environment. • Use of the procedure enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression

  5. Links to Real-world • study of biological processes (ex. biosynthesis of proteins) • localization of gene expression • cell movement • cell fate during development • formation of different organs • screenable marker to identify transgenic organisms

  6. What is transformation? • Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance

  7. ori bla 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

  8. Steps to Make a Transgenic Bacterium • DNA Extraction- get genome from organism containing gene of interest • Splice gene of interest from genome using a restriction enzyme(RE) - RE, chemical that cuts DNA at a recognition site(short sequence of bases) • Splice bacterial plasmid using same RE - plasmid: circular piece of DNA in bacteria video clip

  9. Plasmid may not be exactly circular

  10. Making recombinant plasmid(rDNA) Red= bacteria DNA Blue= human insulin gene

  11. - Details making recombinant plasmid (rDNA) a. Due to sticky ends, gene can be spliced in b. DNA ligase help nuclotides bond • Combined DNA called rDNA or chimera • Put rDNA back into bacteria and grow in culture

  12. Insertion of Gene of Interest into Plasmid

  13. http://www.ncbi.nlm.nih.gov/books/NBK22390/

  14. Using rDNA plasmid to create a transgenic plant

  15. A virus is another biological vector also used to deliver genes

  16. Transgenic Organisms GOAT GOODS. A transgenic goat named Artemis produces in her milk a human-breast–milk compound called lysozyme. Lysozyme destroys bacteria by drilling through their cell walls.E. Scharfen

  17. ara pGLO GFP Amp-r pGLO Plasmid • Ampicillin resistance gene • Green Fluorescent Protein • Aequorea victoria jellyfish gene • ara operon • Regulates transcription of genes in operon and GFP

  18. Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial Transformation

  19. araC pGLO GFP bla Regulating Expression of GFP • Lactose operon • Arabinose operon • pGLO plasmid

  20. lac Operon ara Operon ara Lac Z Y A B A D Effector (Lactose) Effector (Arabinose) ara B A D Lac Z Y A RNA Polymerase B A D Z Y A ara Transcriptional Regulation RNA Polymerase

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

  22. Procedures Transformation Day 1 Day 2

  23. Transformation Procedure • Suspend bacterial colonies in Transformation Solution • Add pGLO plasmid DNA • Place tubes on ice • Heat shock at 42oC and place on ice • Incubate withnutrient broth • Streak plates

  24. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for performing each transformation step? • Transformation solution = CaCl2 Positive charge of Ca+2 ions shields negative charge of DNA phosphates

  25. Cell wall GFP Beta lactamase (ampicillin resistance) Why perform each transformation step? • Incubation on iceslows fluid cell membranes • Heat-shockincreases permeability of cell membrane • Nutrient broth incubationallows beta lactamase expression

  26. What is nutrient broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • carbohydrates • amino acids • nucleotides • salts • vitamins

  27. Volume Measurement

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

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