1. Biotechnology Explorer Program Serious About Science Education

2. 9/11/20152 Aequorea victoria

3. 9/11/20153 Central Framework of Molecular Biology DNARNAProteinTrait

4. 9/11/20154 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. 9/11/20155 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. 9/11/20156 Uptake of foreign DNA, often a circular plasmid What is transformation? GFP Amp Resistance

7. 9/11/20157 A circular piece of autonomously replicating DNA What is a plasmid? Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest ori bla

8. Steps to Make a Transgenic Bacterium 1. DNA Extraction- get genome from organism containing gene of interest 2. Splice gene of interest from genome using a restriction enzyme(RE) - RE, chemical that cuts DNA at a recognition site(short sequence of bases) 3. 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 c. Combined DNA called rDNA or chimera d. 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. 9/11/201517

18. 9/11/201518 pGLO Amp-r GFP ara pGLO Plasmid Ampicillin resistance gene Green Fluorescent Protein Aequorea victoria jellyfish gene ara operon Regulates transcription of genes in operon and GFP

19. 9/11/201519 Bacterial Transformation GFP Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall

20. 9/11/201520 Regulating Expression of GFP Lactose operon Arabinose operon pGLO plasmid pGLO bla GFP araC

21. 9/11/201521 Transcriptional Regulation RNA Polymerase ZYA ZYA Lac Effector (Lactose) ZYA Lac lac Operon BAD ara BAD RNA Polymerase Effector (Arabinose) ara BAD ara Operon

22. 9/11/201522 Gene Regulation ara RNA Polymerase Effector (Arabinose) B B B A A A D D D ara ara Operon RNA Polymerase ara ara GFP Operon GFP Gene ara GFP Gene ara GFP Gene Effector (Arabinose)

23. 9/11/201523 Transformation Procedures Day 2 Day 1

24. 9/11/201524 Transformation Procedure Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42 o C and place on ice Incubate with nutrient broth Streak plates

25. 9/11/201525 Reasons for performing each transformation step?  Transformation solution = CaCl 2 Positive charge of Ca +2 ions shields negative charge of DNA phosphates Ca ++ O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O Ca ++

26. 9/11/201526 Why perform each transformation step?  Incubation on ice slows fluid cell membranes  Heat-shock increases permeability of cell membrane  Nutrient broth incubation allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance)

27. 9/11/201527 Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression carbohydrates amino acids nucleotides salts vitamins What is nutrient broth?

28. 9/11/201528 Volume Measurement

29. 9/11/201529 Grow? Glow? Follow protocol On which plates will colonies grow? Which colonies will glow?