01 BIOTECHNOLOGY

DNA FINGERPRINTING GEL ELECTROPHORESIS Micropipette: Measures solutions by micrometer (  L = 1 millionth of a liter) Use clean pipette tip each time! 1.Calibrate micropipette to 5  L 2.Each person aliquot 5  L of blue colored water (in beaker) onto white paper 3.Calibrate micropipette to 10  L 4.Each person aliquot 10  L of blue colored water (in beaker) onto white paper

DNA FINGERPRINTING GEL ELECTROPHORESIS Gel Electrophoresis Machine: used to filter DNA Fragments by length of strand 1.Calibrate micropipette to 10  L 2.Each person load two gel “wells” with loading dye in the small tube in the green foam 3.Put the top on the machine and plug machine into purple power source 4.When two machines are plugged in, turn the machine to 150V and go back to your desks

Wednesday, February 10 Today: 3 rd /6 th : Finish GATTACA Review Genetic Engineering Techniques 13-2 DNA Fingerprinting Lab Read Pre-Lab Pre-Lab Questions/Practice Tomorrow we will load the machines and run the Gel. We will analyze our results Tuesday. Have a great weekend! Next Week’s Homework: Read/Notes 13-1 by Wednesday Read/Notes 13-3 by Thursday

BIOTECHNOLOGY The manipulation of living organisms or their components to produce useful, usually commercial, products (examples: pest resistant crops, new bacterial strains, or novel pharmaceuticals Topics we will cover: DNA Fingerprinting, Genetic Engineering, Stem Cells, Therapeutic and Reproductive Cloning

13-2 Manipulating DNA New Technologies are used to Extract DNA from cells Cut DNA into smaller pieces Identify the sequence of bases in a DNA molecule Make unlimited copies of DNA Genetic Engineering: making changes in the genetic code of a living organism

13-2 Manipulating DNA DNA Extraction: Chemical procedure to remove the cell membrane and nuclear membrane. Then the DNA is release from the histones that keep it wound in the nucleus.

13-2 Manipulating DNA Cutting DNA: DNA can be cut into smaller pieces by restriction enzymes. Restriction Enzymes break the covalent bonds between nucleotides at specific locations.

13-2 Manipulating DNA Separating DNA: Gel Electrophoresis filters DNA fragments (DNA that has been cut up) through a gel from the negative end to the positive end. Smaller fragments move faster and further through the gel.

13-2 Manipulating DNA Making Copies: PCR (Polymerase Chain Reaction) is a controlled DNA Replication process which can make millions of identical copies of a DNA sample.

13-2 Manipulating DNA Cutting and Pasting: Restriction enzymes can be used to cut portions of DNA out of a sample and a new DNA portion can be added. This combined DNA sample is called recombinant DNA.

DNA Fingerprinting How do biologists get DNA out of a cell? Break down cell membrane and nuclear membrane then unwind DNA from histones. What is a restriction enzyme? A protein that breaks the covalent bonds between nucleotides of DNA at specific locations.

DNA Sample 1 C T T A A G C T T A A G C T T A A G A A G C T T A A G G G C G A A T T C G A A T T C G A A T T C T T C G A A T T C C C G DNA Sample 2 C T T A A G T A C T A C C T T A A G C T T A A G C T T A A G G A A T T C A T G A T G G A A T T C G A A T T C G A A T T C You will be EcoRI, the restriction enzyme that looks for CTTAAG. When it finds this sequence, it cuts between the last A and G. Use your pencil to show these cuts.

Separating DNA 1.Fill in the blanks to outline the steps for gel electrophoresis A mixture of DNA fragments are placed at one end of a porous gel. An electrical current is applied to the gel. When the power is turned on, DNA molecules (fragments) which are negatively charged move toward the positive end of the gel. The smaller fragments move faster.

Gel Electrophoresis Length of Fragment DNA Sample 1DNA Sample

Lane 1: ALane 2: BLane 3: CLane 4: DLane 5: ELane 6: F Crime Scene Suspect 1 Suspect 2

LocusD3S1358vWAFGAD8S1179D21S11D18S51D5S818 Genotype15, 1816, 1619, 2412, 1329, 3112, 1311, 13 Frequenc y 8.2%4.4%1.7%9.9%2.3%4.3%13% LocusD13S317D7S820D16S539THO1TPOXCSF1POAMEL Genotype11, 1110, 1011, 119, 9.38, 811, 11X Y Frequenc y 1.2%6.3%9.5%9.6%3.52%7.2%(Male)

Honors Biology Thursday, February 18 Today you will need 13-1&13-3 notes and your Genetic Engineering Packet. Explain Selective Breeding and how it is used. Explain the process of cell transformation and provide examples of how it is used. Prepare for Transformation Lab. Mrs. Coleman: Room 414

Which trait would you want?

13-1 Changing the Living World Selective Breeding: only allowing organisms with desirable traits to produce the next generation

13-1 Changing the Living World Hybridization: crossing different organisms to combine the desirable traits of each; chromosomes must be close enough for reproduction to take place

13-1 Changing the Living World Inbreeding: continued breeding of organisms with similar desirable traits

13-1 Changing the Living World Genetic variation can be created within a population by inducing mutations. Radiation Chemicals Why would we ever want to do this?

13-1 Changing the Living World …Because sometimes mutants have favorable traits!

13-1 Changing the Living World …Because sometimes mutants have favorable traits! Bacteria that can digest oil. Plants with multiple copies of chromosomes which enhance size and durability. (Polyploidy)

13-3 Cell Transformation Please have 13-3 Notes out in case you need to add anything. Also have the Section 13-3 Review open in your packet. You can probably complete this while we are reviewing! What did Griffith discover? What did Avery discover?

13-3 Cell Transformation During transformation, a cell takes in DNA from outside the cell, which then becomes part of the cell’s DNA. This “new” DNA is called recombinant DNA.

13-3 Cell Transformation Transformation of Bacteria (Prokaryote) Desired gene is located and removed using restriction enzymes. The same enzyme is used to cut bacterial DNA (plasmid) to make room for the new gene. In this example, the gene for human growth hormone is inserted in the bacterial plasmid. The plasmid containing recombinant DNA is returned to the bacteria. **The bacteria’s ribosomes begin expressing the human gene!

13-3 Cell Transformation About plasmids… A plasmid is a circular piece of DNA found in bacteria (prokaryotes). This DNA contains genes necessary for survival. When “new” genes are added to plasmids during transformation, often genetic markers are also added. Genetic markers are used to determine if transformation has occurred or not. Note: The genetic marker we will use in our lab is the gene that should make the bacteria resistant to antibiotics. If this gene is properly transformed into the bacteria, antibiotics will not be able to kill the bacteria…scary!

13-3 Cell Transformation Transformation of Plant Cells (Eukaryote) Examples: genes for insect resistance, frost resistance, and/or drought resistance added to most “edible” plants.

13-3 Cell Transformation Transformation of Animal Cell (Eukaryote) Animal cells may be transformed using a bacteria similar to plant transformation, OR most commonly, desired genes are inserted directly into the nucleus of an egg cell prior to fertilization.

The gene for “bioluminescence” is often used as a genetic marker to see if other genes have been transformed into organisms.

13-3 Cell Transformation So how can transformation be used to help humans? Next I will be giving you a couple of minutes to complete the Section 13-3 Review in your packet.

Genetic Transformation Lab pGlo Objectives: 1.Transform Escherichia coli using the gene for bioluminescence which is naturally found in the jellyfish Aequoria victoria. 2.Determine if transformation was successful using a genetic marker for antibiotic resistance. 3.Observe newly expressed proteins in the transgenic bacteria.

Genetic Transformation The Genetic Creation of Glowing Bacteria Genetic Transformation: Change caused by genes; inserting the genes from one organism into another organism Green Flourescent Protein (GFP): The protein created in the jellyfish when the gene for bioluminescence is expressed What is bioluminescence?

Plasmid: Circular piece of DNA in a bacterial cell; can be passed from one bacteria to another easily; usually contains genes for traits beneficial to survival.

Antibiotic: A compound that blocks the growth and reproduction of bacteria; ampicillin is the antibiotic that should work on E.coli under normal conditions. pGLO: The plasmid that contains the green fluorescent protein gene AND the gene for antibiotic resistance Note: This is a patented product. The company that makes the pGLO plasmid has the legal rights to the product!

Protein Synthesis as it applies to this lab. 1. DNA  RNA  Protein  Genetic Characteristic (Trait) 2. DNA  RNA  Green Fluorescent Protein  Bioluminescence

Lab Materials & Safety

Lab Abbreviations LB (Luria Broth) – Food for the bacteria – carbs! Amp (ampicillin) – antibiotic that kills E.coli under normal conditions Ara (arabinose) – sugar that “turns on” the GFP gene; positive transcription factor pGLO (recombinant plasmid) – contains the antibiotic resistance gene and the gene for bioluminescence

More Lab Materials & Procedures

Genetic Transformation Lab DNA → RNA → Protein → Trait Blue Tube -pGlo (regular bacteria) Orange Tube +pGlo (bacteria with GFP gene and antibiotic resistance gene) TS: Transformation Solution (slightly acidic water) LB: Luria Broth (food) Teacher Has: E.Coli starter plate and pGlo Genetic Transformation Lab DNA → RNA → Protein → Trait

More Lab Materials & Procedures E.Coli TS E.coli pGLO LB TS LB +pGLO -pGLO LB/AMP LB/AMP/ARALB/AMP LB

Petri Dish Explanation of Conditions (Petri Dish Contents) Hypothesize growth on Petri dishes Grow? Glow? Observation of Petri dishes after several days Grow? Glow? +pGlo/LB/Amp GFP & Antibiotic Resistance Gene Luria Broth (bacteria food) Ampicillin (antibiotic) +pGlo/LB/Amp/Ara GFP & Antibiotic Resistance Gene Luria Broth (bacteria food) Ampicillin (antibiotic) Arabinose -pGlo/LB/Amp Regular Bacteria Luria Broth (bacterial food) Ampicillin (antibiotic) -pGLO/LB Regular Bacteria Luria Broth (bacterial food)