Transformation of bacteria with pGLO

Lab 4

pGLO gene

• Bioluminescent jelly fish – Aequorea victoria – GFP causes fish to glow in dark

• Transformed E.coli with GFP will glow a brilliant green color under ultraviolet light

Plasmid

• Circular pieces of DNA – plasmid

• Contain genes for one or more traits – beneficial to bacterial survival

• Transfer plasmids – share these beneficial genes – adapts to new environment – evolution

- Bacterial resistance to antibiotics

pGLO plasmid

• Gene for GFP• Gene for resistance to ampicillin• Operon system – gene on/off regulation

Operon in Prokaryotes

Section that codes for mRNA which later get translated to proteins

Operon in Prokaryotes

• Operator – turn off operon (by regulatory molecule)– Operon get “turned off” b/c RNA polymerase is blocked from

continuing down the strand to the gene– No protein is produced– Blocking and unblocking is how bacteria make certain

proteins certain times– Example: Lactose

Some steps

• Transformation solution – Ca2+ cation of transformation solution neutralizes the repulsive negative charges of phosphate backbone of DNA and phospholipids of cell membrane

• Heat shock – increases permeability of cell membrane to DNA

• Recovery – add LB nutrient broth allows cells to grow and express beta-lactamase – ampicillin resistance protein

• This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis:

• against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and

• against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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