Purpose: To successfully transform E. coli HB101 with the pGLO plasmid.

Hypothesis: If…….then………

Prediction:

Background Information:

*Transformation: The incorporation of “naked” DNA into a cell (usually prokaryote)

Transduction: injection of DNA into a cell using a vector (virus).

Conjugation: the exchange of bacterial DNA between two bacteria using a pilus.

Plasmid: Circular DNA found in bacteria

Example: pUC19, pGLO, etc……

Transformation solution: CaCl₂ is the salt that is used to neutralize DNA molecules

Heat shock: 42 C for 50’. Used to incorporate DNA (plasmid) into bacterial cell

DNA plasmid faces two problems in getting through a cell membrane:

1. Charge of DNA (-). Ca++ neutralizes DNA
2. Size (too large). Heat shock

Gene regulation (operons)

Operon allows for gene regulation in BACTERIA ONLY!!!!

lac-operon

trp-operon

araC-operon (pBAD)

Competent cells

LB medium (Luria Bertoni), both agar and broth.

Transformation Efficiency

Aequorea Victoria (Abstract, extra information/reading)

Genes of interest on the pGLO plasmid

GFP: green fluorescent protein

araC: operator for GFP gene

bla: beta-lactamase (digests ampicillin)