p GLO and Transformation Bioluminescence and GFP Bioluminescence

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p. GLO and Transformation

p. GLO and Transformation

Bioluminescence and GFP • Bioluminescence = the production and emission of light by living

Bioluminescence and GFP • Bioluminescence = the production and emission of light by living organisms – Helps with communication and defense in the biological world • Fluorescence = the emission of light by a substance after it has absorbed light radiation • GFP = green fluorescence protein, produced in certain species of jelly fish, glows green under exposure to blue wavelengths

Aequorea victoria: Source of “glowing gene” for this experiment

Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other Critters http: //www. lafuga. de/GFP_pig. jpg http: //www. technologyreview.

Jellyfish Gene put into Other Critters http: //www. lafuga. de/GFP_pig. jpg http: //www. technologyreview. com/files/21291/monkey_x 600. jpg

Links to the Real World • GFP is a visual marker • Study of

Links to the Real World • GFP is a visual marker • Study of biological processes (ex: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms

Central Framework of Molecular Biology DNA RNA Protein Trait

Central Framework of Molecular Biology DNA RNA Protein Trait

Using GFP as a Biological Tracer http: //www. conncoll. edu/ccacad/zimmer/GFP-ww/prasher. html With permission from

Using GFP as a Biological Tracer http: //www. conncoll. edu/ccacad/zimmer/GFP-ww/prasher. html With permission from Marc Zimmer

Plasmids • Originally evolved by bacteria • Extrachromosomal circular DNA • Often carry genes

Plasmids • Originally evolved by bacteria • Extrachromosomal circular DNA • Often carry genes for antibiotic resistance • Can be passed from one bacterium to another • Can be modified to express proteins of interest http: //www. agen. ufl. edu/~owens/age 2062/On. Line. Biology/OLBB/www. emc. maricopa. edu/faculty/farabee/BIOBK/14_1. jpg

Operons • Systems found in prokaryotic cells that aid in gene regulation • Genes

Operons • Systems found in prokaryotic cells that aid in gene regulation • Genes only get expressed when necessary • Method for controlling transcription/translation – Affects whether RNA Polymerase can bind to the DNA sequence to make m. RNA – Which in turn affects whether m. RNA can be produced to make proteins • Linked genes that help to regulate other genes that code for protein synthesis

3 Parts of an Operon • Promoter = the place where RNA polymerase binds

3 Parts of an Operon • Promoter = the place where RNA polymerase binds • Operator = an “on-off switch” to control transcription of the genes that code for enzymes • Genes = genes that code for enzymes involved in a metabolic pathway

Lac Operon • Inducible operons normally do not allow transcription of the genes—the switch

Lac Operon • Inducible operons normally do not allow transcription of the genes—the switch is off by default • The lac operon: – The enzyme β-galactosidase is required to break down lactose (milk sugar, a disaccharide) into the monosaccharides galactose and glucose – The gene that codes for β-galactosidase is found in the lac operon, along with several other genes that function in lactose metabolism

Lac Operon cont. • Somewhere in the genome is a regulatory gene which produces

Lac Operon cont. • Somewhere in the genome is a regulatory gene which produces a repressor protein – The repressor protein is produced in active form – It therefore immediately binds to the operator, switching the lac operon off – This prevents transcription of the genes involved in lactose metabolism • The presence of lactose allows the operon to be turn on – Lactose binds to the repressor altering its shape so it detaches from the operator – Allows RNA polymerase to bind and transcribe the genes for making the enzymes necessary for digesting lactose

Arabinose Operon • Works similar to the Lac operon, in the presence of arabinose

Arabinose Operon • Works similar to the Lac operon, in the presence of arabinose the operon is turned on and genes for making arabinose digesting enzymes are sequenced • If you add the gene for making glowing proteins next to the genes for making the enzymes to digest arabinose you will also get p. GLO proteins

Transformation • Transformation = the process in which foreign DNA gets introduced into a

Transformation • Transformation = the process in which foreign DNA gets introduced into a cell • Transformation occurs naturally in the wild during conjugation

Transformation cont. • Transformation can be performed in a laboratory setting my manipulating the

Transformation cont. • Transformation can be performed in a laboratory setting my manipulating the environment of the cell and introducing DNA thru a vector (a DNA molecule that gets used to carry the foreign genetic material

Reasons for Each Transformation Step 1. Incubation on ice slows fluidity cell membranes 2.

Reasons for Each Transformation Step 1. Incubation on ice slows fluidity cell membranes 2. Use of Ca. Cl 2 helps the plasmid vector get incorporated into the bacterial genome because positive Ca ion attract the negatively charged DNA 3. Heat-shock increases permeability of cell membrane 4. Nutrient broth incubation allows beta lactamase expression

Selection for plasmid uptake • Antibiotic becomes a selecting agent – only bacteria with

Selection for plasmid uptake • Antibiotic becomes a selecting agent – only bacteria with the plasmid will grow on antibiotic (ampicillin) plate all bacteria grow only transformed bacteria grow a a a LB plate a a a LB/amp plate cloning

Bacterial Transformation The uptake of DNA Bacterial Cell Chromosomal DNA Plasmids

Bacterial Transformation The uptake of DNA Bacterial Cell Chromosomal DNA Plasmids