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Biotechnology Explorer Program Serious About Science Education Aequorea victoria 5/23/2017 2 Central Framework of Molecular Biology DNA 5/23/2017 RNA Protein Trait 3 5/23/2017 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 4 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 5/23/2017 5 What is transformation? Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance 5/23/2017 6 What is a plasmid? A circular piece of autonomously replicating DNA ori bla Originally evolved by bacteria May express antibiotic resistance gene or be modified to express proteins of interest 5/23/2017 7 Steps to Make a Transgenic Bacterium 1. 2. 3. 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 Plasmid may not be exactly circular Making recombinant plasmid(rDNA) Red= bacteria DNA Blue= human insulin gene - 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 Insertion of Gene of Interest into Plasmid http://www.ncbi.nlm.nih.gov /books/NBK22390/ Using rDNA plasmid to create a transgenic plant A virus is another biological vector also used to deliver genes 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 5/23/2017 17 pGLO Plasmid Ampicillin resistance gene Green Fluorescent Protein Aequorea pGLO victoria jellyfish gene ara ara Amp-r GFP operon Regulates transcription of genes in operon and GFP 5/23/2017 18 Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA pGLO plasmids 5/23/2017 Beta lactamase (ampicillin resistance) 19 Regulating Expression of GFP Lactose operon araC Arabinose operon pGLO bla pGLO 5/23/2017 GFP plasmid 20 Transcriptional Regulation lac Operon Lac Z Y ara Operon A ara B A D Effector (Lactose) Lac Z Y A Effector (Arabinose) ara B RNA Polymerase Z 5/23/2017 Y A A D RNA Polymerase ara B A D 21 Gene Regulation ara GFP Operon ara Operon ara B A D ara Effector (Arabinose) Effector (Arabinose) ara B A D ara RNA Polymerase ara 5/23/2017 B A D GFP Gene GFP Gene RNA Polymerase ara GFP Gene 22 Transformation Procedures Day 1 Day 2 5/23/2017 23 Transformation Procedure 5/23/2017 Suspend bacterial colonies in Transformation Solution Add pGLO plasmid DNA Place tubes on ice Heat shock at 42oC and place on ice Incubate with nutrient broth Streak plates 24 Reasons for performing each transformation step? Ca++ O Transformation solution = CaCl2 Ca++ O P O Base O CH2 O Sugar Positive charge of Ca+2 ions shields negative charge of DNA phosphates O Ca++ O P O Base O CH2 O Sugar OH 5/23/2017 25 Why perform each transformation step? Incubation on ice slows fluid cell membranes Cell wall GFP Heat-shock increases permeability of cell membrane Nutrient broth incubation allows beta lactamase expression 5/23/2017 Beta lactamase (ampicillin resistance) 26 What is nutrient broth? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression 5/23/2017 carbohydrates amino acids nucleotides salts vitamins 27 Volume Measurement 5/23/2017 28 Grow? Glow? 5/23/2017 Follow protocol On which plates will colonies grow? Which colonies will glow? 29