cloning and biotech 2

Plasmid

A small circular double-stranded DNA molecule that replicates independently of the bacterial chromosome

Origin of replication (ori)

A DNA sequence that allows a plasmid to be copied inside a host cell

Copy number

The number of plasmid molecules present in a single cell

Selectable marker

A gene (e.g. antibiotic resistance) that allows identification of cells containing a plasmid

Ampicillin resistance (ampR)

A selectable marker that allows only plasmid-containing bacteria to survive on ampicillin

Multiple cloning site (MCS)

A short DNA region containing many restriction enzyme sites for inserting DNA fragments

Blue-white selection

A screening method that distinguishes plasmids with inserts from those without inserts

lacZ gene

Encodes β-galactosidase and is disrupted when DNA is inserted into the MCS

White colony

Indicates successful insertion of DNA disrupting the lacZ gene

Blue colony

Indicates an empty plasmid with an intact lacZ gene

Shuttle plasmid

A plasmid that can replicate in more than one type of host organism

Ti plasmid

A natural shuttle plasmid from Agrobacterium tumefaciens used to transfer DNA into plants

Artificial chromosome

A vector designed to carry very large DNA fragments

Yeast artificial chromosome (YAC)

A vector capable of cloning DNA fragments up to ~2 Mb

Bacteriophage vector

A virus-based vector used to clone medium-sized DNA fragments in bacteria

Phage λ vector

A bacteriophage vector with an insert capacity of approximately 9–25 kb

Transformation

The process of introducing plasmid DNA into bacterial cells

Heat shock transformation

A method that uses temperature change to allow plasmid entry into bacteria

Electroporation

A transformation method that uses an electric pulse to introduce DNA into cells

Reporter gene

A gene whose product is easily detected to confirm gene expression or insertion

GFP

A reporter gene that produces green fluorescence when expressed

pET expression system

A plasmid system designed for high-level protein expression in E. coli

T7 promoter

A strong viral promoter used in pET vectors that requires T7 RNA polymerase

T7 RNA polymerase

A viral RNA polymerase required to transcribe genes under a T7 promoter

DE3 strain

An E. coli strain engineered to produce T7 RNA polymerase

IPTG

A lactose analog used to induce expression of genes under lac operon control

Inducible expression system

A system where gene expression can be turned on by adding a specific molecule

Cloning strategy

A planned approach to isolate, insert, transform, and select a DNA fragment in a vector