1 / 26

Transforming E. coli with a Recombinant Plasmid

Transforming E. coli with a Recombinant Plasmid. Laboratory 5. Starting new here!!!!!. Experiments do not rely on past results. Overview . Purpose: Get the pARA-R plasmid containing the rfp gene into bacterial cells

asher-jacobson
Download Presentation

Transforming E. coli with a Recombinant Plasmid

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. Transforming E. coli with a Recombinant Plasmid Laboratory 5

  2. Starting new here!!!!! Experiments do not rely on past results

  3. Overview • Purpose: • Get the pARA-R plasmid containing the rfp gene into bacterial cells • Get those cells to express the rfp gene and make the mutant fluorescent protein

  4. Introduction • Transformation • Process of taking up foreign pieces of DNA • In this case, a plasmid • Usually happens through conjugation • Not very common in mature! • Can occur under experimental conditions (1 cell in a thousand!) • Advantages include antibiotic resistance • All cells that undergo binary fission after insertion of plasmid will possess it:

  5. Plasmid DNA Insertion

  6. Transgenic Colony Allowed to Grow

  7. Introduction • Transformation efficiency: • Size of plasmid • Large • Less likely to take up plasmid • Must pass through plasma membrane and cell wall • Small • More likely to pass through • Shape of plasmid • Supercoiled easiest to pass through • Nicked-circle or mulitimer harder to get through • Most likely your tubes will contain

  8. Introduction • What are competent cells?!?! • Cells ready to receive plasmids through lab procedures • Soaked in calcium chloride • PM and DNA negatively charged • Calcium ions (Ca++) neutralize charges to allow for plasmid to pass through PM • Need to “heat shock” cells • Creates pressure differences • Cold then hot • Then feed and recover!!

  9. Calcium ions Preparing competent cells for transformation Bruce Wallace Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  10. Competent Cells pARA-R Transforming Escherichia coli with pARA-R Bruce Wallace Recombinant Plasmids

  11. Calcium ions pARA-R Transforming Escherichia coli with pARA-R Bruce Wallace Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  12. Introduction • Spread cells on various plates (3) • LB plate • Only bacterial food • LB/amp plate • Contains ampicillin • Antibiotic prevents bacteria from forming CW • Cells that contain ampr produces protein that destroys ampicillin • Cells will grow! • LB/amp/ara plate • Contains arabinose • Needed to express the rfp gene • If take up plasmid, this helps with transcription of gene

  13. Materials • Reagents and Cultures • pARA-R tube • 100 µL competent cells (LMG) • 350 µL LB broth (sterile) • Crushed ice in Styrofoam cup • Sterile agar plates • LB • LB/amp • LB/amp/ara • Equipment and Supplies • P-20 micropipette and tips • P-200 micropipette and tips • 42oC water bath • 1 pack sterile spreaders • Plastic microfuge tube rack • 1.5 mL microfuge tubes • Marking pens • Disinfectant spray • Cybersafe

  14. What will you need to do? • Discuss proper aseptic techniques • Protect themselves • No contamination! • Assign tasks…. • Turn on water bath the day before to check temperature

  15. What will you need to do? • KEEP COMPETENT CELLS FROZEN until transformation day • Day of lab: • Take out number of tubes need for transformation • Each group will need 100 µL of cells • Each tube contains @ 500 µL of cells • Place tubes in wet ice to defrost • Will take about 10-15 minutes • Resuspend cells prior to aliquoting • Gently pump in and out with P-200 pipette • Return any unused tubes with cells immediately to Styrofoam chest and place into freezer

  16. What will you need to do? • Cells need to be in contact with ice or water during exercise • Push tubes to bottom of foam rack • Have cell-contaminated waste bag • Deposit tips, tubes, and spreaders that have come in contact with bacteria • Bag will be autoclaved after use

  17. Methods….diluted version • 2 clean microfuge tubes – KEEP PIPETTE TIPS SEPARATE!!!! • P+ • P- • Label BOTTOM of plates (agar side) near edges • After step 12, let rest for a few minutes at room temperature • Step 14d • Clamshell opening • GLIDE spreader, do not dig • Be sure to invert the plates when incubate • Decrease condensation and skewed results

  18. Predictions…… P+ P+ P- P- P+ LB LB/amp LB/amp/ara

  19. Predictions…… P+ P+ P- P- P+ + + + + - LB LB/amp LB/amp/ara

  20. Conclusions • 2 tubes • P+ contains E. coli and plasmid • P- contains only E. coli

  21. Growth of transformed bacteria on various plates Bruce Wallace P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

More Related