Plasmid Cloning

What is DNA cloning?

the process of making multiple, identical copies of a particular piece of DNA known as recombinant DNA

How is recombinant DNA made?

inserting a gene or any other DNA fragment of interest into a plasmid

What is a plasmid?

small DNA molecules located outside the chromosome that replicate independently

What can plasmids function as in DNA cloning?

vectors to transfer genetic material

What are the parts of a plasmid?

promoter, the target gene, antibiotic resistance gene

What is the end goal of DNA cloning?

to have the recombinant DNA of a plasmid placed inside a bacteria be replicated and passed down to offspring

Describe what happens in the first steps of cloning DNA.

restriction enzymes cut matching sequences of both the plasmid and the target gene; target gene is sealed to plasmid with DNA ligase

How does a restriction enzyme split the target gene?

it recognizes a restriction site

How can DNA ligase seal the backbone between a target gene's single-stranded overhang with a matching plasmid's single cut site?

it catalyzes a reaction with ATP to create a link between the phosphate group at the 5' end of one DNA strand and the hydroxyl group on the 3' prime end of the other

What are other, unusable products that can form during ligation?

sealed plasmids without the target gene, or plasmids with the target gene inserted backwards

How is foreign DNA introduced into bacteria?

through transformation, heat shock allows for pores to form in the bacteria's membrane which makes it easier for the DNA to enter

What is the purpose of a plasmid's antibiotic resistance gene?

to help the bacteria survive when placed in an environment with antibiotics

Why would scientists place bacteria in an environment (ex. petri dishes) with antibiotics?

to encourage the bacteria to take in the recombinant DNA

What can bacteria colonies containing the appropriate plasmid be turned into?

plasmid factories or protein factories

What is the purpose of plasmid production?

to produce more of the recombinant DNA for further experimentation, or to create more complex plasmid systems

How is a protein factory created?

growing a bacteria colony into a large culture

How can a protein factory be utilized?

a chemical signal can induce the reproduced gene to begin protein production in mass amounts

How are proteins obtained from the bacteria?

lysing, or slicing open, the bacteria

What is affinity chromatography?

a method of separating all the bacteria's molecules from the target protein

Why is affinity chromatography essential?

to purify proteins before use

How does affinity chromatography single out a protein?

by coating the extracted molecules in an antibody that only the target protein can bind to, that protein will remain in the container as the excess molecules are washed away