Ch 18: Bacterial Transformation

What does genetic transformation mean?

change caused by genes, involving the insertion of a gene into an organism in order to change the organism’s trait

Where is Green Fluorescent Protein (GFP) sourced and what trait does it bring about?

• source = bioluminescent jellyfish Aequorea victoria

• causes the jellyfish to fluoresce and glow in the dark

What are plasmids?

• smaller, circular pieces of DNA separate from the one large chromosome bacteria typically have

• typically contain genes for one or more traits that are beneficial for bacterial survival

Can plasmids be shared? What are real-life implications of this?

• yes, bacteria CAN transfer plasmids back and forth

• this allows bacteria to adapt to new environments → bacterial resistance to antibiotics is due to the transmission of plasmids

List and explain the components of pGLO plasmid.

• ori (origin of replication)

• araC gene regulation system (creates AraC regulatory protein, which controls the expression of the GFP in transformed cells)

• P _BAD promoter (AraC binds here to prevent transcription)

• GFP gene

• bla gene (for B-lactamase, which allows resistance to the antibiotic ampicillin)

How is the GFP gene “switched on” in transformed cells?

• by adding the sugar arabinose to the cells’ nutrient medium

• arabinose alters the structure of AraC, allowing RNA pol to bind to P _BAD

• transformed cells will appear white on plates not containing arabinose, and fluorescent green when arabinose is included in nutrient agar medium

How are cells that have been transformed selected?

through growth on antibiotic plates

So which component of the transformed cell is doing the glowing?

A) cytoplasm

B) expressed protein

C) cell membrane

B) expressed protein (GFP specifically) is what’s causing the bacteria to glow

What was the point of adding CaCl₂ into each tube at the beginning?

the Ca²⁺ neutralizes the negative charges on both the bacterial cell membranes & DNA, reducing repulsion and allowing the plasmids to sit near the bacterial surface

What does the heat shock step do? Why is there a strict time constraint?

• it temporarily increases membrane permeability & drives DNA into the cell

• if you go too short → membrane doesn’t become permeable enough & you get low transformation efficiency

• if you go too long → proteins start to denature & cell gets stressed/dies

What are the conditions of the four plates we tested? What results did we get for each plate?

• LB/amp & +pGLO = growth but no glow

• LB/amp/ara & +pGLO = growth AND glow

• LB/amp & -pGLO = no growth

• LB & -pGLO = lawn of growth but no glow

What is transformation efficiency? How is it calculated

• indication of the extent to which E. coli becomes genetically transformed

transformation efficiency = total number of GFP cells on LB/amp/ara plate /

amount of DNA spread on agar plate