1 / 26

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?.

kanan
Download Presentation

Transformation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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! 

More Related