GENETIC ENGINEERING BIOLOGY 2/2A MOTZKO

Flavr Savr (1994) Combined tomato antisense (mutated) DNA to prevent the ripening process

GENETIC ENGINEERING  Recombinant DNA = utilizing DNA from two different sources  Transgenic DNA = utilizing DNA from two different species  Hence, all transgenic DNA is recombinant but not all recombinant DNA is transgenic

Fast Growing Salmon (2010) Combines eel pout and salmon DNA to produce fish that grows 2x fast

A Cleaner Pig Sty (2012) Combines E.coli, mouse and pig DNA to produce swine that produce 70% less phosphorus waste

And A Less Gassy Cow (2009) Bioengineered cows produce 25% less methane gas as a byproduct of rumination

Venomous Cabbage (2002) Combines scorpion and cabbage DNA to create vegetable that is poisonous to caterpillars but not to humans

A Banana A Day… Banana saplings infected with virus carrying DNA codes for immunity to Hepatitis B. Vaccine then passed to humans via eating bananas

The Incredible, Edible Egg (2007 ) Bioengineered British chickens lay eggs that contain monoclonal antibodies used to fight arthritis and multiple sclerosis

Evergreen (or not so green) (2003 ) Colorado State bioengineers evergreens that change color when under attack from insects or when environmentally stressed.

Biosteel (2000) University of Wyoming Study combines DNA of orb-weaving spiders with goats to produce large quantities of spider-silk in lactating females.

Fluorescent Feline Clones (2007) South Korean experiment seeks to utilize bioluminescence to show that gene expression can be regulated. If so, felines could be created that express human diseases for research purposes.

WHAT?  A bacterial transformation  Forcing a bacterial cell to transcribe and translate a sequence of foreign DNA into protein

WHY?  To create a transgenic organism (contains DNA from two different species)  To show how genes are organized, controlled and expressed  To demonstrate how artificial selection affects the evolution of a population

WHO?  Bacteria: Escherichia coli HB101  Source of Foreign DNA: Aqueoria victoria (naturally bioluminescent jellyfish)

Aequorea victoria

Why Escherichia coli?  Prokaryotic  No introns  Rapid growth  Non-pathogenic

HOW?  The jellyfish gene for bioluminescent protein (GFP) has already been spliced into a ring of DNA called a plasmid  E.coli forced to absorb these plasmids through a combination of chemicals and heat shock

Contents of pGLO Plasmid  Amp = gene coding for beta lactamase (makes E.coli HB101 resistant to the antibiotic ampicillin)  araC = gene coding for arabinase, an enzyme that allows E.coli HB101 to metabolize the sugar arabinose  GFP = gene that codes for Green Fluorescent Protein

pGLO Operons  Beta Lactamase = codes for an enzyme that breaks down the antibiotic ampicillin  Arabinase = codes for enzyme which helps hydrolyze the sugar substrate arabinose

How Do We Get The pGLO Plasmid Into E.coli HB101 ?

Four Testing Groups (Control & Independent Variables)  Four Test Groups Plates  1 LB (luria broth)  2 LB + Ampicillin (antibiotic)  1 LB + Ampicillin + Arabinose (sugar)

Contents of Each Vial  Vial 1 (for LB – and LB/amp-) gets 1 loop of bacteria, transformation solution but NO pGLO  Vial 2 (for LB/amp + and LB/amp/ara +) gets 1 loop of bacteria, transformation solution and pGLO

Transformation Solution  Bacterial plasma membrane will have its semi- permeability altered through use of dilute CaCl 2 solution

Heat Shock  42 degrees C for 50 seconds, then back to ice

Luria Broth & Plating  Supply cells with Luria broth for recovery from stress  After recovery, pour half of DNA – vial onto LB- and half onto LB/amp –  Pour half of DNA + vial onto LB/amp+ and half onto LB/amp/ara+ plate

Incubation  Incubate at enteric (37 C) temperatures for hours

HOW IS OUR GENOME A BUREAUCRACY?

Operon System  A series of structural and regulatory genes arranged in a manner such as to produce various proteins only when needed by the cell  A more efficient way to arrange and manage gene expression  Only 3-5% of genes need to be expressed at any given time

Operon Components  Promoter Region = Place where RNA polymerase binds to DNA strand to start the transcription of genes into mRNA  Operator: Location adjacent to promoter where repressor protein binds, blocking RNA polymerase from binding to the promoter  Structural Genes : The genes that are transcribed when RNA polymerase can bind to the promoter region  Repressor Gene : codes for repressor protein to block the operator region

Inducible System  Inducible System: structural genes can only be transcribed when a specific substrate is present  Ex: Lactose operon (Jacob & Monod)

What Is Required To Induce Each Operon To Code For Its Protein?

Operon Function  When the substrate is absent, the repressor protein is produced and blocks access to the promoter of the operon  When the substrate is present, the repressor is inhibited and the unblocked promoter can be accessed

If Function Dictates Form, How Can Recombinant DNA Be Expressed ?

Antibiotic Selection Process  Not all bacteria will absorb the plasmids. We will kill off those that fail to absorb the plasmids by placing some of bacteria in environments containing the antibiotic ampicillin.  Those bacteria that survive must have picked up the pGLO plasmid because it contains the gene beta- lactamase, which destroys the antibiotic.

Inducing GFP Production  Not all the bacteria that have absorbed the pGLO plasmid will produce GFP.  To induce/force the bacteria to produce GFP, they must also be forced to produce the enzyme arabinase.  Why? Because the GFP gene was inserted into the plasmid adjacent to the arabinase gene.  If you “turn on” the arabinase gene, you will “turn on” the GFP gene by default.

Transformation Efficiency  Def: How many E. coli cells picked up the pGLO plasmid relative to the amount of pGLO plasmid to which they were exposed (expressed in trasnformants/µg)  Calculate transformation efficiency for LA+ and LAA+ plates (as they were the only ones exposed to plasmid)  Use pp in your pGLO manual