LO 3: Plasmids 101

3 mechanisms for DNA to be introduced into non-daughter cells

1. Conjugation (bacterial sex between friends) 2. Transduction (DNA carried by a viral vector) 3. Transformation: artificially pushing DNA (like a plasmid vector) into a bacterium using CaCl2 + heat shock or electricity (electroporation)

What is an origin of replication for? What would happen to a plasmid if it didn't have it?

The origin of replication in a plasmid allows the plasmid to replicate on its own. Without it, the plasmid wouldn't be able to replicate on its own.

Can plasmids be lost altogether from a cell? If so, how could it happen?

Yes, through a process called curing. This can happen when there is no selection pressure to maintain the plasmid.

Name the kinds of genes found on naturally occurring plasmids

1. Conjugation: tra genes (etc.) to facilitate bacterial sexual exchange of genetic material (between neighbors) 2. Alternative Physiological Functions: a. the ability to live on an alternate food source b. ability to degrade toxins such as herbicides + camphor, octane, etc. c. ability to resist death from metal ion poisoning (Co, Ni, Cd, Zn) 3. Antibiotic Resistance: the ability to resist or degrade specific antibiotics 4. Virulence Plasmids: host cell invasion, produce hemolysin, tumorigenicity in plants

3 basic elements for a plasmid to be used as a cloning vector

1. Contain genes controlling their own replication 2. If conjugative, they contain transfer (tra) functions that facilitate the process 3. They are stably maintained in the host of choice

What is a nucleosome?

146 bp DNA + histone core

What are chromatin?

DNA wrapped in histones (used for silencing DNA during transcription)

Describe when a plasmid replicates inside a cell

Gram-negative bacteria: initiation at the origin of replication and bidirectional replication around the circle. Some have unidirectional replication. Gram-positive bacteria: replication by the rolling circle mechanism, giving a single strand intermediate, with a protein that binds to the 5' end, which primes DNA synthesis

Are plasmids only passed on to daughter cells, or can they be passed on to neighbors?

Plasmids can be passed onto neighboring cells via conjugation, which is controlled by the tra region in the parent plasmid

How quick do plasmids replicate?

Plasmids replicate quickly compared to chromosomal replication (10th or less of total time for chromosomal replication)

Compare and Contrast a plasmid with a bacterial chromosome

Size: For the most part, Bacterial DNA is much larger than plasmids (1 kb to >1 mb; about 5% the size of a chromosome) Shape: Plasmids do not have an extracellular form and are circular (some are linear). Genes: Plasmids are double-stranded like chromosomes. Essential Genes: Plasmids carry unessential (yet helpful) genes. Bacterial chromosomes carry essential genes.

Explain (a) part of what determines how many copies and (b) what kinds of plasmids can be found inside one cell.

A: Copy number is controlled by genes on the plasmid and by interactions between the host and the plasmid B: -Resistance Plasmids -Toxin Encoding Plasmids -Bacteriocins -Engineered Plasmid -Episomes -F Plasmids -Conjugative Plasmids