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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 Explorer Program Serious About Science Education
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. • Use of the procedure enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression
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
What is transformation? • Uptake of foreign DNA, often a circular plasmid GFP Amp Resistance
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
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
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 • Combined DNA called rDNA or chimera • Put rDNA back into bacteria and grow in culture
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
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
Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial Transformation
araC pGLO GFP bla Regulating Expression of GFP • Lactose operon • Arabinose operon • pGLO plasmid
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
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
Procedures Transformation Day 1 Day 2
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
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
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
What is nutrient broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • carbohydrates • amino acids • nucleotides • salts • vitamins
LB/Amp LB/Amp/Ara LB Grow? Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow?