Biotech Labs: Discover Genetic Engineering Endeavors

1. Animations • Nova Videos • Journey into Human DNA interactive:http://www.pbs.org/wgbh/nova/body/journey-into-human-dna.html • Cracking the Code of Life: http://www.pbs.org/wgbh/nova/genome/program.html • Miracle of Life: http://www.pbs.org/wgbh/nova/miracle/program.html • Personal Genome Project: http://www.pbs.org/wgbh/nova/body/church-genome-vid.html • How to Sequence DNA (complex): http://www.pbs.org/wgbh/nova/body/sequence-DNA-for-yourself.html • Epigenetics (13 min): http://www.pbs.org/wgbh/nova/body/epigenetics.html • Gene Switches Interactive: http://www.pbs.org/wgbh/nova/body/gene-switches.html • Stem Cells: http://www.pbs.org/wgbh/nova/body/stem-cells-breakthrough.html • Tay Sachs (one wrong letter) – from the “Cracking the Code of Life” video • http://www.pbs.org/wgbh/nova/genome/program.html • Animations on DNA Sequencing, DNA Extraction, Cloning, Gel Electrophoresis, loading a gel, etc are • all available on the Amgen Biotech site: • http://www.amgenbiotechexperience.com/curriculum/curriculum-resources • Biotechnology Overview with a lot of good info: http://biotechnology.amgen.com/index.html- timeline: http://biotechnology.amgen.com/timeline.html- need to make a HW on this

2. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 1.1: Learn how to use a pipette to dispense small volumes of solutions • Lab 1.2: Learn how to use the technique of gel electrophoresis to separate molecules based on their size • Lab 2: Learn how to use restriction enzymes to cut DNA at specific locations • Lab 3: Learn how to use DNA Ligase to join together DNA fragments • Lab 4: Learn how to use gel electrophoresis to check to verify successful creation of a plasmid with the desired genes • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene (that is, insert a desired gene into bacteria so you can grow it to produce the desired protein)

3. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 1.1: Learn how to use a pipette to dispense small volumes of solutions

4. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 1.1: Learn how to use a pipette to dispense small volumes of solutions • Lab 1.2: Learn how to use the technique of gel electrophoresis to separate molecules based on their size Negatively charged molecules (like DNA) are attracted to the positive electrode. Smaller fragments move through the porous gel faster than larger fragments.

5. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 1.1: Learn how to use a pipette to dispense small volumes of solutions • Lab 1.2: Learn how to use the technique of gel electrophoresis to separate molecules based on their size

6. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 2: Learn how to use restriction enzymes to cut DNA at specific locations.

7. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 2: Learn how to use restriction enzymes to cut DNA at specific locations.

8. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 2: Learn how to use restriction enzymes to cut DNA at specific locations

9. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 2: Learn how to use restriction enzymes to cut DNA at specific locations. Cut two genetically engineered plasmids into DNA fragments that contain specific genes of interest: From the Plasmid pKAN-R • red fluorescent protein (rfp)– the protein we want to grow and isolate • promotor (pBAD)– site at which RNA polymerase binds to DNA to initiate transcription (an RNA copy of the genes are made which then moves to the ribosomes which use them to build the proteins) From the plasmid pARA • ampicillin resistance gene (ampR)– enables us to selectively grow only bacteria that have the plasmids with the desired genes in them • arabinose activator gene (araC) – codes for a protein that blocks the promoter unless the sugar Arabinose is present to remove it (thus the genes are only able to be activated and their proteins made when arabinose is mixed in) Restriction Enzymes being used to make the cut: BamH I and Hind III

10. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 3: Learn how to use DNA Ligase to join together DNA fragments Join together the DNA fragments produced in Lab 2, resulting in the production of different plasmids with various combinations of genes:

11. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 3: Learn how to use DNA Ligase to join together DNA fragments Join together the DNA fragments produced in Lab 2, resulting in the production of different plasmids with various combinations of genes:

12. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 4: Learn how to use gel electrophoresis to check to verify successful creation of a plasmid with the desired genes

13. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 4: Learn how to use gel electrophoresis to check to verify successful creation of a plasmid with the desired genes

14. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 4: Learn how to use gel electrophoresis to check to verify successful creation of a plasmid with the desired genes

15. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene 1) Insert plasmid with desired gene into E coli bacteria 2) Verify that it worked by growing the bacteria in the presence of ampicillin (which acts as an indicator for transformation) and arabinose (which is required to activate the rfp gene)

16. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene 1) Insert plasmid with desired gene into E coli bacteria

17. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene 2) Verify that it worked by growing the bacteria in the presence of ampicillin (which acts as an indicator for transformation) and arabinose (which is required to activate the rfp gene)

18. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene 2) Verify that it worked by growing the bacteria in the presence of ampicillin (which acts as an indicator for transformation) and arabinose (which is required to activate the rfp gene)

19. Amgen Biotech Labs Overview In this set of labs, students will: • Lab 5: Learn how to transform competent cells with a plasmid containing a desired gene 2) Verify that it worked by growing the bacteria in the presence of ampicillin (which acts as an indicator for transformation) and arabinose (which is required to activate the rfp gene)

20. Amgen Biotech Labs Introduction Questions • Genetic Engineering is the branch of biology that uses special procedures and techniques to change an organism’s DNA.One example is removing DNA from one organism and inserting it into another organism.

21. Amgen Biotech Labs Introduction – Answers to Pre-lab Questions • Genetic Engineering is the branch of biology that uses special procedures and techniques to change an organism’s DNA. • Insulin is the hormone responsible for regulating glucose levels in the blood • Record all observations, procedures, and data in a lab book • Gene cloning can be used to treat genetic diseases in which a person’s body is unable to make a needed protein, or the protein does not work well, or enough of the protein is not made due to mutations in the gene for the protein or in genes that control expression of the protein. Gene cloning enables us to make the protein artificially by using “good” copies of the gene inserted into bacteria that produce the protein. • The two discoveries in the 1970’s and 1980’s that made this possible are

22. Polymerase Chain Reaction (PCR) • Used to synthesize many copies of a segment of DNA from very little source DNA http://www.dnalc.org/resources/animations/pcr.html

23. Polymerase Chain Reaction (PCR) Method used to copy DNA • Strands are “melted” apart using heat • Primers and nucleotides (NTP’s) are added and the mix is gradually cooled • Results in formation of complementary strands • Cycle is repeated, doubling number of DNA strands each time http://www.dnalc.org/ddnalc/resources/pcr.html http://www.sumanasinc.com/webcontent/animations/content/pcr.html

24. Gel Electrophoresis • Separates proteins or fragments of DNA based on size • Electricity moves molecules from – towards + electrodes • Gel retards progress so that gradient can be observed • A marker is used for size comparison • A stain is used to make proteins/DNA visible http://www.dnalc.org/ddnalc/resources/electrophoresis.html

25. Gel Electrophoresis • Proteins must be coated in SDS to give them a uniform negative charge • Different types of gel are used to separate different substances • Agarose gel (used for DNA) • Polyacrylamide gel (for proteins)

26. DNA Fingerprinting using Gel Electrophoresis • DNA is cut up using restriction enzymes • Results in different sized pieces determined based on the genetic variation between individuals Restriction Enzymes cut DNA at specific sequences, forming fragments

27. DNA Fingerprinting using Gel Electrophoresis • DNA is cut up using restriction enzymes • Results in different sized pieces determined based on the genetic variation between individuals • Fragments are run on a gel via gel electrophoresis • Forms a pattern of bands unique to each person

28. Selective Breeding • Crossing organisms that have desirable traits with the goal of obtaining offspring with those traits

29. Genetic Engineering • Create new combinations of genetic material • Transfer genes between different members of the same species • Transfer genes between different species • Create new genes by recombining sections from other genes • Gene segments code for protein domains • Protein domains have specific functions within the protein • Protein domains are conserved among various “related” proteins

30. Recombinant DNA Technology Restriction Enzymes cut DNA at specific sequences, forming fragments restriction sites Sticky ends are where hydrogen bonds have been cleaved leaving behind unmatched bases that are looking to form new H-bonds

31. Recombinant DNA Technology Restriction enzymes can be used to cut out specific regions of a chromosome containing a gene of interest. This fragment can then be inserted into another strand of DNA using ligase. Recombinant DNA – a chromosome, plasmid or other segment of DNA that contains nucleotide sequences from more than one source

32. Recombinant DNA Technology • Plasmids • Circular strands of DNA in addition to cell’s chromosomes • Contain only “nonessential” genes • Toxins • Antibiotic resistance • Conjugation pili • Found only in prokaryotes • Are copied at the same timeas the chromosomes prior tobinary fission (reproduction) • Can be easily transferred from one cell to another

33. Recombinant DNA Technology • Gene for desired protein cut out of chromosome using a restriction enzyme • Same restriction enzyme is used to cut open a plasmid (cloning vector) • DNA segment is inserted into the opened plasmid creating a recombinant plasmid • Recombinant plasmid is inserted into a bacterium and allowed to reproduce and synthesize the desired recombinant protein

34. Recombinant DNA Technology Bacteria containing recombinant DNA can be used to manufacture synthetic protein products called Biologics (for example, insulin and human growth hormone) Bacteria are grown in vats. As they grow and divide, they produce the proteins from genes inserted into their genomes along with their normal proteins. The synthetic proteins are purified from the batch. Biologics are strictly regulate by the FDA just as pharmaceuticals are.

35. Recombinant DNA Technology • A gene from Bacillus thuringiensis for a natural insecticide can be inserted into: • Corn plants directly (genetically modified food), or • Pseudomonas fluorescens, a different bacteria that grows on corn plants Recombinant DNA can be inserted directly into cells or be delivered via bacteria or viruses. New variations of plants and animals can be created using these genetic engineering techniques. Transgenic Organisms - plant, animal, or bacteria that has been genetically modified to contain a gene from a different species

36. Recombinant DNA Technology • Genetic engineering is used to modify foods in order to: • Increase crop yield • Increase shelf life • Increase market desirability (color, texture, flavor, size, etc) • Increase nutritional value • Resist weather variables (freezing, drought, etc) • Resist crop pests and diseases Recombinant DNA can be inserted directly into cells or be delivered via bacteria or viruses. New variations of plants and animals can be created using these genetic engineering techniques. Transgenic Organisms - plant, animal, or bacteria that has been genetically modified to contain a gene from a different species

37. Recombinant DNA Technology Agrobacterium tumefaciens is used to transfer recombinant genes into plants via the Ti plasmid Recombinant DNA can be inserted directly into cells or be delivered via bacteria or viruses. New variations of plants and animals can be created using these genetic engineering techniques. Transgenic Organisms - plant, animal, or bacteria that has been genetically modified to contain a gene from a different species

38. Recombinant DNA Technology Pseudomonas putida contains genes for degrading oil which are being recombined into other species in order to develop bacteria for bioremediation of oil spills Recombinant DNA can be inserted directly into cells or be delivered via bacteria or viruses. New variations of plants and animals can be created using these genetic engineering techniques. Transgenic Organisms - plant, animal, or bacteria that has been genetically modified to contain a gene from a different species

39. Recombinant DNA Technology Used first time in 1990 to treat severe combined immunodeficiency syndrome (SCID) Recombinant DNA can be inserted directly into cells or be delivered via bacteria or viruses. Gene Therapy - the insertion of genes into an individual's cells and tissues to treat a disease

40. Cloning • Remove nucleus from donor egg • Insert nucleus from individual you are attempting to clone into the donor egg (or fuse the cells together) • Add chemicals to induce mitosis (send fertilization signal and other growth signals) Reproductive Cloning – goal is to make an entire cloned organism Therapeutic Cloning – goal is to create embryonic stem cells that can be used to grow replacement tissues

41. Stem Cells Stem cells are cells that can divide and grow to form multiple different types of cells based on the chemical instructions they receive from other cells • Can be artificially directed to form tissues • Embryonic (totipotent) stem cells have the potential to become any tissue type • Adult stem cells have the potential to become any tissue type in their cell lineage(Ex: hemapoetic cells can become red blood cells or one of many types of white blood cells) Differentiated cells are mature cells that have achieved their final state (Ex. a red blood cell)

43. The Human Genome Project • International effort to sequence the entire Human Genome • Coordinated by the U.S. Department of Energy and the National Institutes of Healthhttp://www.ornl.gov/sci/techresources/Human_Genome/home.shtml • Began in 1990, Completed in 2003 • Next steps: • Figure out where genes start and stop • Figure out which proteins each gene codes for • NOVA: Cracking the Code of Lifehttp://www.pbs.org/wgbh/nova/genome/program.html http://www.pbs.org/wgbh/nova/genome/sequ_flash.html

44. Web Resources • McGraw Hill’s Biotechnology Animations • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html • PCR • http://www.youtube.com/watch?v=ZmqqRPISg0g • http://www.dnalc.org/resources/animations/pcr.html • Gel Electrophoresis • http://www.dnalc.org/ddnalc/resources/electrophoresis.html • http://www.dnalc.org/resources/animations/gelelectrophoresis.html • http://learn.genetics.utah.edu/content/labs/gel/ • Recombinant DNA • http://www.bioteach.ubc.ca/TeachingResources/Applications/GMOpkgJKloseGLampard2.swf • http://webapps.css.udel.edu/biotech/rDNA.html • Stem Cells • http://www.sumanasinc.com/scienceinfocus/sif_stemcells.html • http://www.dnalc.org/resources/animations/stemcells.html • Human Genome Project • http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml • http://www.pbs.org/wgbh/nova/genome/program.html • http://www.pbs.org/wgbh/nova/genome/sequ_flash.html