Chapter 2: Plasmids and Phages

Plasmid

Extrachromosomal circles of self-replicating DNA

In bacteria or yeast

Episomes

plasmids that can integrate into the genome

pGlo

Plasmid containing the GFP sequence and ampicillin resistance gene

Selectable Marker

Gene used to detect the Plasmid's Presence, normally an anti-biotic resistance gene

bla/Apr

Ampicillin Resistance Gene

Selectable Marker for pGlo

Ori

Origin of Replication

araC

where arabinose is present it codes for a transcription factor which allows GFP Transcription

Why have araC

Plasmid is integrated and replicated before being activated to prevent the bacteria from destroying it

Cloning Site/Polylinker

Site on the plasmid with multiple Resitriction Sites, used to add DNA

Copy Number

The number of plasmids in a cell

Inverse to plasmid size

Integration

Episome is added to the chromosome

Plasmid Incompatability

Bacteria can only take up one of a type of plasmid so we know that all of the plasmids in it are clones

PuC19 Plasmid

Contains APR and lacZ alpha, MCS in lac Z

Colonies are blue if gene was not inserted, white if it was

PMAL-C6T Plasmid

Has Bla

Lac operon and repressor are present

IPTG keeps lac operon turned on

Bacteriophage

Virus that inject their DNA into Bacteria

Consist of DNA and Protein

M13

Filamentous Phage with a lysogenic life cycle, used in DNA sequencing and mutagenesis

M13 genome size

3 kb

M13 DNA

ssDNA

Replicative Form (RF)

M13's ssDNA is converted into double stranded DNA in a host cell ad acts like a plasmid

M13's Life Cycle

1. Enters via pilus

2. ssDNA gets a second strand and turns into the Replicative Form

3. DNA is copied using the bacterial DNA Polymerase to create more ssDNA and Transcribed to create viral proteins

4. New Viruses assemble and exit the cell

Plaques

Clearings on a bacterial lawn where viruses have killed the bacteria

M13 as a Plasmid

Has viral genes, ori, and lac Z alpha as an indicator

Can see if gene was taken up by plaque color

Lambda Phage

Head and tail phage, used to create large libraries due to having a large genome, Can be Lyctic or Lysogenic depending on the environment

Library

Collection of captured DNA that represents a genome

Lambda DNA

Mostly double stranded, ends are sticky and complementary so it can form a loop

lyctic cycle for Lambda

DNA is injected, replicates and creates proteins

New viruses assemble and rupture the cell

Lysogenic Cycle for Lambda

DNA is injected, circularizes, joins the host chromosome and stays dormant while the cell divides

When the environment changes, the Viral DNA excises itself, makes new viruses, and ruptures the cell

Rolling Replication

Used by Lambda

Genome length units called Catenane are formed and separated by cos sites

The cos sites are cut by an endonuclease so the DNA can enter a capsid

Lambda Genome Size

48.5 kb

Packaging Limit

Lambda Protein Capsids will not pack genomes that are too big or small

(Under 37 or above 52 kb)

Lambda vs M13 use

Lambda: large genome to make large libraries

M13: small genome to make ssDNA, plasmids, and small libraries

Baculovirus

Used to create large amounts of proteins in moth cells

Gemini Virus

Proposed viruses to use for plant genetic engineering

SV40 and Adenovirus

Used for gene therapy and designing cell lines in mammals