Aim: What are plasmids?Insulin is a hormone (Protein) produced by cells in the pancreas, it causes cells in the liver, skeletal muscles, and fat tissue to absorb glucose from the blood.Except in the case diabetes mellitus insulin is constantly made in the body to remove excess glucose from the blood, which otherwise would be toxic.

Do now: Do you know how Synthetic insulin is made? If not write down 3 theories on how it is made in the lab

VocabularyPlasmidEukaryoticBacteriaCompetent Calcium Chloride

Ori geneSelectable markerAntibiotic resistanceAmpicilin Luciferase

What is a plasmid?A plasmid is a DNA molecule that is separate

from, and can replicate independently of, the chromosomal DNA.

They are double stranded and, in many cases, circular.

Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (cells contain a nucleus and other structures enclosed within membranes)

How can plasmids be used? A plasmid can be used as a vehicle that can

carry artificially inserted DNA. It will replicate in E. coli, and when E. coli

replicates.In a way you can see a plasmid as a tiny DNA

factory.

Why use E.coli?Easy and relatively inexpensive to grow in

the labCan easily be made competent (able to

transform) using simple chemicals such as Calcium Chloride

How can plasmids enter bacteria?Plasmids enter the bacterial

cell very easily. This happens naturally and

may account for the rapid spread of antibiotic resistance in hospitals and elsewhere.

Plasmids can be introduced into bacteria in the laboratory by making cells competent.

Why are plasmids important?Plasmids are extremely

valuable tools in the fields of molecular biology and genetics, specifically in the area of genetic engineering.

They play a critical role in such procedures as gene cloning, recombinant protein production (e.g., of human insulin), and gene therapy research.

How are plasmid made?DNA workers have engineered a number of

features into many plasmids that make them easier to use. First the essentials:

1. it must have the “ori” gene, this gene enables the plasmid to be replicated in the host cells

2. it must have a "selectable marker“ this makes it so that only cells that have the plasmid will grow in culture. Usually and antibiotic resistance gene is used.

What plasmid will we be using?pVIBResistant to the antibiotic ampicillin (ampr)Contains gene for synthesizing Luciferase

a bioluminescence enzyme found in fireflies. The name is derived from Lucifer, the root of which means 'light-bearer' (lucem ferre)

Plasmid DNA as vaccines for Cancer and Infectious DiseasesThere is a concept of

plasmid DNA to be used a a protective vaccine strategy for cancer and infectious diseases.

However, the crossover application into human studies has been met with poor results based on the DNA vectors inability to provide clinically relevant therapeutic benefit