Lab 8 Warm-Up What are plasmids? What gene(s) are on the plasmid being used in this lab? How can we know if cells have been successfully transformed in this lab?

Warm-Up (Lab 8 Review) What are the 3 genes found on the pGLO plasmid? What is the main function of each gene? How can you tell if the E.coli bacteria successfully picked up the pGLO plasmid? If the goal was to produce as much GFP as possible, what type of plate would you grow the E.coli on (what substances would be in the agar on the plate)?

Bacterial Transformation AP Biology Investigation 8

Transformation Uptake of foreign DNA from surroundings Plasmid: small ring of DNA that carries a few genes Antibiotic resistance (eg. ampicillin) AP Lab 8: transform pGLO plasmid into E.coli bacteria

pGLO Plasmid Beta Lactamase (bla): ampicillin resistance Green Fluorescent Protein (GFP): jellyfish gene, glows under UV light araC Regulator Protein: regulates GFP transcription; activated by presence of arabinose (sugar)

Transformation Lab Luria Broth (LB) = liquid nutrients for bacterial growth Transformation Solution (TS) = CaCl2, neutralize DNA backbone and phospholipids and allows DNA to enter cells Heat Shock: increases permeability of cell membrane to DNA +pGLO = solution contains pGLO plasmid -pGLO = no pGLO plasmid

Predictions: Plate 1: LB.amp +pGLO Plate 2: LB/amp/ara +pGLO Plate 4: LB –pGLO Which plates will have bacterial growth? Which plates will have bacteria that will GLOW?

Lab Instructions Read instructions CAREFULLY Use sterile technique with petri dishes, pipettes and innoculating loops Use a NEW pipette or loop for each specimen Using pipettes: 100µl, 250 µl markings Safety first: gloves, goggles, biohazard containers Keep specimens ON ICE Timing is Important! Step #8: Heat Shock for 50 seconds Step #10: Close tubes and flick to mix bacteria suspension before plating

Observations Draw what you see for each plate. Plate 1: LB.amp +pGLO Plate 2: LB/amp/ara +pGLO Plate 3: LB/amp –pGLO Plate 4: LB –pGLO Count and record the number of colonies visible on each plate. Note any other observations (color, etc.)

Data Analysis: Calculating Transformation Efficiency Total # colonies on plate/amt. DNA spread (μg) Transformation efficiency (TE) is a way to describe how effective you were at getting a plasmid into bacteria. The protocol we used generally has a TE between 8.0 x 102 and 7.0 x 103 transformants per microgram of DNA

Factors that affect transformation efficiency Plasmid size (large vs. small) Forms of DNA (supercoiled vs. relaxed DNA) Growth of cells (competent cells) Transformation protocol (heat shock time, temp., growth media, chemicals, contaminants) Damage to DNA