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Making Cells Glow: Bacterial Transformation with pGLO Plasmid DNA

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Making Cells Glow: Bacterial Transformation with pGLO Plasmid DNA

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  1. Making Cells Glow:Bacterial Transformation with pGLO Plasmid DNA

  2. Bacterial Transformation,Genetic Engineering,and Recombinant Proteins

  3. Essential Components of Genetic Engineering bacterial A TRANSGENEintended to give the host cell or organism new or altered traits. animal • A VECTORa plasmid or virus DNA used to • assemble the recombinant construct • maintain it in its temporary and permanent host cells • introduce the transgene into cells • ensure expression of the transgene in the new host cell HOST CELLSwith their own genomic DNA plant

  4. Plasmids are frequently used as vectors. plasmids host chromosome • Extrachromosomal bacterial DNAs • Small • 4000 bp; compared to bacterial chromosome of ~4 million base pairs • Easy to work with • Can be removed, altered, then returned to cells. • Replicate independently of bacterial chromosome • Single or multiple copies per cell • Discovered in nature as a source of antibiotic resistance Some plasmids integrate into the host genome http://commons.wikimedia.org/wiki/File:Plasmid_episome.png

  5. Features of a TypicalCloning Vector reporter gene polylinker • Origin of Replication • ensures replication of DNA in a host cell • Selectable Marker • allows for selection of transformants; usually confers antibiotic resistance • Reporter Gene • gene whose phenotype changes depending on whether a foreign transgene has been inserted into the plasmid • Promoter(s) • promotes transcription of selectable marker and transgene/reporter gene • Polylinker or Multiple Cloning Site (MCS) • series of closely spaced, unique restriction sites at which the plasmid can be cut (linearized) to allow insertion (ligation) of the transgene into the plasmid promoters origin of replication selectable marker

  6. bla: a common selectable marker Ampicillin Beta-Lactamase • An antibiotic • Prevents the growth of bacteria by inhibiting an enzyme that is needed for building new cell wall peptidoglycan • Chemical structure based on a Beta-lactam ring • Beta-lactam antibiotics include penicillins (amoxycillin) and cephalosporins • An enzyme • Chemically breaks the beta-lactam ring, inactivating the enzyme • The bla gene • Encodes the beta-lactamase enzyme • Makes bacteria resistant to ampicillin (ampr)

  7. Issues in Moving TransgenesBetween Hosts Prokaryotes Eukaryotes • Bacteria • Simple cell structures • No nucleus; DNA spread throughout the cell • Simple gene structures • Protein-coding DNA sequence (open reading frame, or ORF) is contiguous • No machinery for RNA splicing • Simple promoters • Fewer transcription factor proteins • Fungi, Protists, Plants, Animals • Complex cell structures • Internal membrane-enclosed organelles, including a nucleus • Complex gene structures • Protein-coding DNA sequence (exons) is interrupted by non-coding sequences introns • Requires RNA splicing to convert pre-mRNA (primary transcript; exons + introns) into mRNA (exons) • Complex promoters • Many transcription factor proteins

  8. Eukaryotic Gene Structure http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif

  9. cDNA Cloning of Eukaryotic Genes into Prokaryotic Hosts • Since eukaryotic genes have introns that prokaryotic cells can’t remove, a cDNAtransgene is created from a DNA copy of the mRNA with introns removed. • cDNA: complementary DNA • Transgene must be attached to a prokaryotic promoter to ensure transcription in new bacterial host.

  10. Regulation of Transcription in Prokaryotes at the Para and Plac Promoters • In the absence of inducer (lactose or arabinose), transcription is turned OFF • Repressor protein binds to the operator, blocking the RNA polymerase from the promoter. • In the presence of inducer (lactose or arabinose), transcription is turned ON • Inducer binds to the repressor protein, causing a change in its shape. The repressor falls off the operator, allowing RNA polymerase to bind to the promoter and transcribe the gene.

  11. Recombinant Proteins • Proteins that are produced through genetic engineering. • Encoded by the introduced transgene. • Produced upon the transgene’s transcription and translation. • Can be purified from the transgenic cells or organisms. • Can be produced in much higher quantities that protein available from natural sources.

  12. Examples of Recombinant Proteins Medicine Industry & Consumer Products • Human insulin (1982): Used to treat diabetes. • rHGH or human growth hormone (1985): Stimulates growth (height) and development of muscle. • Cadaver-derived natural HGH transferred Creutzfeldt-Jacob disease (1985) • rBST or bovine somatotropin(1994): Stimulates milk production in cows • Required or permissible labeling of rBST milk or non-rBST milk is debated • EPO or erythropoeitin: Stimulates creation of red blood cells. • Used to treat anemia in cancer chemotherapy patients. Common blood doping agent for athletes. • tPA or tissue plasminogen activator: Enzyme given to heart attack patients to dissolve blood clots blocking arteries. • Factor VIII: Blood clotting factor missing in hemophiliacs • In Laundry Detergents • Protease for proteins, lipases for greases, and amylases for carbohydrates • Amylases and Maltases • For production of high fructose corn syrup from corn starch • Cellulases and Ligninases • Enzymes that digest cellulose into sugars to be fermented in ethanol for biofuels • Pectinases • Clarify fruit juices • Rennin • Used in cheese production

  13. Session 1:Transforming the Bacteria Mixing bacterial cells and DNA under transformation conditions. Introduces DNA into cells.

  14. araCgene: encodes the repressor protein that blocks transcription at Para promoter in absence of arabinose The pGLOPlasmid Origin of replication Para promoter: allows transcription of the gfp gene when cells are treated with arabinose bla gene: a selectable marker; encodes beta-lactamase enzyme; confers ampr phenotype gfpgene: a transgene; encodes Green Fluorescent Protein (GFP); confers glowing phenotype

  15. Label Your Transformation Culture Tubes • Use a lab marker to label two 15ml round-bottom culture tubes: • -DNA & your initials • +DNA & your initials • Place these tubes in your ice cup to chill. • It is very important that the transformation reactions be keep cold. Don’t handle these tubes or have them out of the ice for more than a few seconds at a time. +DNA initials -DNA initials

  16. Making the Transformation Mixtures • To each tube, add 100µl of competent E. coli cells. • Provided in a microtube in your ice cup • Pipet slowly (the cells are fragile) • Carefully deposit the drop of cells to the very bottom of the tube. • Keep tubes on ice. • Promptly replace the snap-on caps to avoid contamination by bacteria and fungi in the air. • Don’t forget: always use a fresh pipet tip each time! • To the –DNA tube, add 10µl of TE Buffer directly to the drop of cells • To the +DNA tube, add 10µl of 5ng/µl pGLO plasmid DNA directly to the drop of cells. 100µl cells 100µl cells +DNA initials +DNA initials -DNA initials -DNA initials 10µl TE 10µl DNA

  17. Cold-Incubating the Transformation Mixtures • Gently tap the bottom of each tube to gently mix the cells and solutions. • Incubate on ice for 15 minutes. • During this time, DNA becomes attached to the outer surface of the cells.

  18. Heat-Shocking the Transformation Mixtures • Bring your ice cup with the two culture tubes to the 42°C heat block. • Quickly place your pair of tubes into the heat block. Note the time. • After exactly 45 seconds, quickly remove your pair of tubes and immediately place them back in your ice cup for at least one minute. • This “heat-shock” step opens pores in the cell’s membranes, allowing the DNA to enter some cells. The heat shock requires instantaneous transitions between cold to hot to cold.

  19. Initial Cell Culture:Recovery and bla Gene Expression • Add 800µl of LB broth to each culture tube. • Don’t forget: always use a fresh pipet tip each time! • Replace the caps promptly to avoid contamination. • Tap the bottom of the tube to mix. • Place the tubes into the foam adapter mounted on a vortex mixer. • Your samples will be agitated at room temperature for about 45 minutes. This gives the new genes (on the plasmid DNA) introduced into the cells time to be transcribed and translated into proteins. +DNA initials -DNA initials 800µl LB broth 800µl LB broth

  20. Teacher Note • After about a class period of incubation (40-50 minutes), transfer the tubes to a lab refrigerator (without food!) for overnight storage.

  21. Session 2:Spread-Plating the Transformation Cultures Growing the transformation cultures on non-selective, selective, and indicator plates.

  22. Culture Media • LB • Luria-Bertani medium: a rich medium that provides a complete mixture of nutrients (sugars, amino acids) and vitamins in which bacteria can grow. • agar • a substance added to media that makes it semi-solid

  23. Culture Media Additives • amp: ampicillin • an antibiotic that kills bacteria, except those cells that contain genes that provide resistance (such as the beta-lactamase or bla gene, sometimes called an ampicillin-resistance or ampr gene) • selective medium • a growth medium that causes the death, or prevent the growth, of some cells but not others • ara: arabinose • a sugar that induces transcription of a gene by removing the repressor protein from the gene’s specific “ara” promoter • indicator medium • a growth medium that causes some cells to appear differently than other cells, indicating the presence or absence of certain traits

  24. Why Grow Transformation Cultures On Selective Media? • The transformation process is very inefficient. • Only a tiny fraction of the cells actually take up the DNA. • We face a “finding a needle in a haystack” problem. • How do we detect, and obtain, only the cells that have been successfully transformed (the “transformants”)? • “Burn down the haystack!” • Kill off all the non-transformants on selective media. • Cells lacking the pGLO plasmid will lack its bla gene, and thus will be sensitive to ampicillin.

  25. To possess a trait, you need to both possess the gene and express it. • The gfp gene encodes the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoreavictoria. • For a cell to have GFP, it must transcribe and translate the gfp gene. • Arabinose induces the transcription of the gfpgene. • The gfp gene is expressed when transformed cells are treated with arabinose. http://en.wikipedia.org/wiki/File:Aequorea_victoria.jpg http://en.wikipedia.org/wiki/File:GFP_structure.png

  26. Labeling the Plates Keep plates agar-side UP! Non-selective plate -DNA +DNA +DNA -DNA -DNA +DNA LB amp ara LB LB amp LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __ Selective plate Selective & Indicator plate

  27. Spread-Plating the Transformation Cultures One partner does these three. The other partner does these three. 200µl 200µl -DNA -DNA -DNA +DNA +DNA +DNA LB amp ara LB LB amp LB amp ara LB amp LB Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __ 200µl 200µl +DNA initials -DNA initials 200µl 200µl

  28. Spread-Plating • Your foil packet contains two sterile yellow spreaders. Feel the foil packet and find the end shaped like a triangle. Carefully open the foil at the stick (not the triangle) end, keeping the triangle ends covered with foil. Keep the spreader in the opened pack for now. • Turn your three –DNA plates over (agar side on bottom) and apply 200µl of your –DNA culture to each of the three –DNA plates: LB, LB amp, and LB amp ara. Use a fresh pipet tip each time. • Remove one spreader from the pack (keep the other spreader covered) and use it to gently spread the liquid across the entire surface of each plate, turning the plate as you spread. Don’t press too hard, or the agar will tear. Place the used spreader in the collection bin. • Repeat using the other spreader to apply the +DNA culture to each of the three +DNA plates.

  29. Incubating Your Cultures • Allow your plates to sit, agar side down, for a few minutes to allow the liquid to absorb into the agar. • Tape your set of six plates together using colored lab tape. • Label the tape with your class period. • Place you set of six plates into the 37°C incubator for an overnight incubation.

  30. Predict Whether Cells Will Grow On Each Plate, and What They Will Look Like

  31. Teacher Note • After overnight incubation, if the students will not be observing their results the following day, wrap the plates in parafilm and store them in a refrigerator (with no food!).

  32. Session 3:Interpreting Results Examining for evidence of transformation and recombinant gene expression.

  33. Use Your “Two Minds” Imagining Mind Thinking Mind • Imagines what’s possible • Finds all alternatives • Decides what’s real • true or false • Eliminates alternatives

  34. Scientific Thinking is Critical Thinking E + A = C Evidence“Facts we SEE” Assumptions“Things we THINK” Conclusions“Claims we MAKE” • Materials • Procedures • Experimental Design • CONTROLS • Observations • Data • Results

  35. Words of Wisdom from Sherlock Holmes • “It is an old maxim of mine that when you have excluded the impossible, whatever remains, however improbable, must be the truth.”

  36. Designing An Experiment:Experimental Variables Manipulated Variables Controlled Variables Responding Variables Uncontrolled Variables

  37. Manipulated Variables • Also called the Independent Variable. • The condition or treatment that is changed or manipulated during the experiment. • Each sample is subjected to different conditions for the manipulated variable: treatment, amount, time, duration, etc. • The manipulated variable is the "cause" for which we wish to identify an "effect".

  38. Controlled Variables • Conditions and treatments that are identical for all samples within the experiment • Conditions that are to be ruled out as affecting the outcome.

  39. Responding Variables • Also called the Dependent Variable. • The properties to be observed or measured. • The "effect(s)" associated with changes in the manipulated variable.

  40. Uncontrolled Variables • Factors which may impact experimental samples or subjects differently, resulting in effects that are not due to the manipulated variable. • Experimenter error • Bias • Environmental conditions • Non-random sampling

  41. Analysis of Results

  42. Arrange your plates like this. -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  43. What do you conclude from THIS plate ALONE? What ELSE might you conclude? +DNA +DNA -DNA +DNA -DNA -DNA LB amp LB LB LB amp ara LB amp LB amp ara Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __ Critical Thinking involves identifying and considering ALL alternatives! Negative results have no meaning EXCEPT in comparison to a POSITIVE CONTROL.

  44. What do you conclude from THIS plate ALONE? -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  45. What is the Manipulated Variable? Controlled Variables? Responding Variable? Conclusion? -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  46. What is the Manipulated Variable? Controlled Variables? Responding Variable? Conclusion? -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  47. What is the Manipulated Variable? Controlled Variables? Responding Variable? Conclusion? -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  48. Examining for Production of Green Fluorescent Protein • Turn out the room lights. • Hold the UV lamp over your plates. • Do not look directly into the UV lamp. • Record which of your plates have colonies that glow green.

  49. What is the Manipulated Variable? Controlled Variables? Responding Variable? Conclusion? -DNA +DNA -DNA -DNA +DNA +DNA LB amp ara LB amp LB LB amp ara LB LB amp Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Team ___or initials Period __ Period __ Period __ Period __ Period __ Period __

  50. Good Experimental Design Seeks to control variables. Confirms all of our assumptions about materials and procedures. Allows us to conclude a clear CAUSE-and-EFFECT relationship between the MANIPULATED VARIABLE and the RESPONDING VARIABLE.