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Transformation

Transformation. AP Big Idea #3: Genes and Information Transfer connected with AP Big Idea #1 (Evolution) & #2 (Cellular Processes). How has our understanding changed?. What distinguishes stem cells from differentiated cells?.

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Transformation

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  1. Transformation AP Big Idea #3: Genes and Information Transfer connected with AP Big Idea #1 (Evolution) & #2 (Cellular Processes)

  2. How has our understanding changed?

  3. What distinguishes stem cells from differentiated cells?

  4. What types of genes are turned on/off as the path towards differentiation occurs?

  5. Its all about regulation! • Gene Regulation in Bacteria • Operons • Use of bacterial gene regulation systems in biotechnology • Gene Regulation in Eukaryotes: (We’ll come back to this in a later powerpoint!!!) • Regulation at the DNA Level • Regulation at the transcription level • Regulation at the translation level

  6. Bacterial Operons • Operon: set of genes that control the production of a certain protein product needed by the cell under specific conditions. • Inducible • Repressible • What do you think is the difference between these?

  7. Inducible: Example LAC

  8. How does the presence of lactose “induce” transcription?

  9. Repressible: TRP operon

  10. Why are operons necessary? • What is the advantage of E. coli having evolved? • A lac operon? • A trp operon?

  11. Using Bacterial Genetics for Biotechnology • Transformation: • Bacteria can uptake eukaryotic DNA and become gene factories.

  12. What is a plasmid?

  13. Basic Structure of a Plasmid

  14. What does a plasmid do in a bacteria? “Sex”

  15. Selectable markers • Resistance to antibiotics (like kanomycin) • If bacteria gets plasmid; it will grow in presence of antibiotic. • Screens out bacteria that don’t receive plasmid.

  16. Origin of Replication • Bacteria divide rapidly to form colonies. • They must be able to replicate the plasmid into their daughter cells.

  17. Making a plasmid: • Restriction enzymes: • Enzyme present in bacteria that cuts DNA at certain points (used to protect bacteria from viruses) • Used in biotechnology to cut open a plasmid and glue in a gene of interest • Ligase: • Enzyme used to glue in inserted piece of DNA (where did we talk about this enzyme before?)

  18. Problems that can be encountered

  19. The hard part is done! • We bought pre-engineered pGLO plasmid. • Plasmid was engineered and then copied by BioRAD!

  20. Engineering a bacteria

  21. Steps we will do: • Heat shock bacteria to add plasmid. • Grow (clone) bacteria to give them a chance to replicate plasmid (E. coli populations double every 20 minutes) • Incubate bacteria and allow them to produce protein=green fluorescence protein • Use an operon to turn on/off the presence of protein production!

  22. pGLO Lab: • How can we alter the transformation process to improve transformation efficiency? • Each lab group

  23. What you must know before the lab: • Research the pGLO plasmid. Be able to identify: • The operon, the selectable marker, the inserted gene. • Read through the manual (available on BioRad’s website and through a quick web search…pGLO student manual) • Predict what will happen on each plate you make: • LB -pGLO • LB/Amp -pGLO • LB/Amp +pGLO • LB/Amp/Ara +pGLO

  24. Lab Day: • Decide how will your group “tweak” the procedure to attempt to increase transformation efficiency. • Identify the expected result for each plate and understand why each control is used. • Identify in your lab notebook how you will calculate transformation efficiency (for your plate that produces GFP) the next day.

  25. Data and Analysis Day: • Calculate control transformation efficiency. Put in spreadsheet (we will have 3 sets of data for class control) • Calculate the average transformation efficiency for your 3 experimental titles. • In your groups, make sure you understand the specific purpose of your group’s lab!

  26. Follow-up: • This lab is a model of using transformation to produce a protein product. • Find a real life example of how bacterial transformation was used (or how GFP was used) and explain understanding this model is useful (implications and connections!) • Remember…we are using Semester 2 Lab rubric…Here is a chance to show the skills you learned as a big lab group! 

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