1- Plasmids

What are plasmids

Plasmids are extrachromosomal molecules of DNA, most being double stranded close circular molecules

What is an origin of replication (ori)

Unique DNA sequence inside a plasmid that allows them to use the host cell’s replication machinery to replicate. This replication is not usually tied to host chromosome replication

Can there be multiple types of plasmids inside the host?

Yes, as long as the plasmids are compatible with each other and with the host

What is a non-integrative plasmid

When bacterial host cell divides, plasmids non-equally “divy up” into the daughter cells

What is an episome

A plasmid that that is included in the chromosome for cell division. The plasmid can come out of the integrated cell (equal plasmid division)

What is conjugation

The transfer of plasmid DNA between a bacterial donor and recipient. The cells attach via a pilus (a hollow appendage present on the donor cell). A copy of the plasmid is then passed to the recipient cell

What is the starting point of conjugation replication

Origin of transfer (orit)

Use of plasmids in bacteria

Can carry genes beneficial to the host, such as antibiotic resistance genes, metabolic enzymes, or virulence genes

What is a virulence gene

A gene that provides some pathologic capability to the bacteria. Ex = E.coli EAE gene to break down intestinal cells

What is a protein expression plasmid

Protein expressions (has RNA polymerise bind to make proteins, and it has poly-A proteins) - used for recombinant hormones, insulin, IL-2

4 basic features of a recombinant plasmid

• Origin of replication (ori)

• Selectable marker/antibiotic resistance gene

• Multiple cloning site

• Insert

How can antibiotic resistance be used as a selectable marker

When bacteria are grown on agar containing the antibiotic, only bacteria that have taken up the plasmid survive and form colonies

What is a multiple cloning site (MCS)

Short segment of DNA containing several unique restriction enzyme sites that allow insertion of foreign DNA

Use of cutting a plasmid with multiple restriction enzymes

Cuts at 2 points so we can DNA insert with directionality

What is the plasmid insert

Foreign DNA cloned into the multiple cloning site (MCS) for further study. Insert may be a gene, promoter, regulatory sequence, or other DNA fragment

What is a promoter

A promoter is an upstream (of MCS) DNA sequence that recruits RNA polymerase and initiates transcription. It helps drive transcription of inserted target gene

What is a primer

Short, single stranded DNA sequence that provides a starting point for PCR amplification or DNA sequencing

Use of universal primer-binding sites

Can be used in plasmids to amplify or sequence across an insert to verify the recombinant plasmid