Do Now What is E.coli? And why is it used for biotech? What have you learned so far in this biotech unit?

What is the evolutionary purpose for plasmids? Do Now What is the evolutionary purpose for plasmids? What are aseptic techniques?

Objectives SWBAT: Using the biotechnology skills developed so far, 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 Explain the necessary biological processes to make this change happen

Transforming E. coli with a Recombinant Plasmid Laboratory 5

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:

Plasmid DNA Insertion

Transgenic Colony Allowed to Grow

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

Introduction What are competent cells?!?! Need to “heat shock” 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!!

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

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

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

Introduction Spread cells on various plates (3) LB plate LB/amp 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

Materials Reagents and Cultures Equipment and Supplies 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

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

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

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

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

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

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

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

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