selectable marker

Selectable Marker

Definition: This element is required for the maintenance of the plasmid in the cell. Due to the presence of the selective marker, the plasmid becomes useful for the cell. Under the selective conditions, only cells that contain plasmids with the appropriate selectable marker can survive. Commonly, genes that confer resistance to various antibiotics are used as selective markers in cloning vectors. The drawbacks of this aproach are: 1. loss of selective pressure as a result of antibiotics degradation and inactivation. 2. contamination of the product or biomass by antibiotics, which may be unacceptable from medical or regulatory considerations.

Frequently Used Antibiotic Agents
(Mechanism of Action)

Experimental Tips for Antibiotics Use

Alternative Selection Strategies

Inspect Commercially Available Plasmids

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Alternative Selection Strategies

The following reviews address this issue:

Swartz JR., Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol 2001;12:195-201 - read the last part of the section: "Low gene dosage"
Baneyx F., Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 1999 ;10:411-21 - read the section: "Plasmid copy number and maintenance".

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Frequently Used Antibiotic Agents
Antibiotic	Type of Cell	Mechanism of Action	Mechanism of Resistant Gene
Ampicilin	Prokaryote	Binds to and inhibits a number of enzymes in the bacterial membrane that are involved in the synthesis of the cell wall.	The amp^rgene on the plasmid codes for an enzyme that is secreted into the periplasmic space of the bacterium, where it catalyzes hydrolysis of the B-lactam ring of ampicillin, with concomitant detoxification of the drug.
Chloramphenicol	Prokaryote	Binds to the ribosomal 50S subunit and inhibits bacterial protein synthesis.	The cam^rgene encodes for a tetrameric, cytosolic protein (MW of each subunit 23,000) that in the presence of acetyl coenzyme A, catalyzes the formation of hydroxyl acetoxy derivatives of chloraphenicol that are unable to bind to the ribosomes.
Kanamycin	Prokaryote	Binds to ribosomal 70S subunit and inhibits bacterial protein synthesis.
Tetracyclin	Prokaryote	Binds to a protein of the 30S subunit of the ribosome and inhibits ribosomal translocation	The tet^r gene encodes a membrane associated protein that prevents the antibiotics from entering the cell.
Blasticidin S	Prokaryote & Eukaryote	Inhibits protein synthesis.	The blasticidin^r gene encodes for a blasticidin deaminase, which converts blasticidin S to a non toxic deaminohydroxy.
Neomycin	Eukaryote	Binds to the 30S and in some cases the 50S subunit causing miscoding; inhibits initiation and elongation during protein synthesis.	The neo^r gene encodes neomycin phosphotransferase which confers resistance to G418 (Geneticin), and the gene, encoding dihydropholate reductase (DHFR). When DHFR is used, the recipient cells must have a defective DHFR gene, which makes them unable to grow in the presence of methotrexate (MTX), unlike transfected cells with a functional DHFR gene
Hygromycin B	Eukaryote	Inhibits protein synthesis by disrupting translocation.	The hyg^r gene - hygromycin B phosphotransferase (HPH) -

Sigma's List of Antibiotic sorts several antibiotics according to their mechanism of action.

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Experimental Tips for Antibiotics Use

When choosing a plasmid with a certain antibiotic resistence, make sure this resistence is not already conferred in the host cell. Otherwise it cannot serve as a means of selection for the plasmid's presence (bacterial strains for Dna propagation & cloning, bacterial strains for protein expression).
If the chosen vector is intended for use in eukaryotic cells, make sure you have two antibiotic selectable markers: 1. for eukaryotes 2. for prokaryotes, so you can multiply the vector in bacterial cells. (Use the appropriate selectable marker for each host!)
The use of ampicillin as a selectable marker is somewhat problematic. Amp^r cells secrete the enzyme B-lactamase into the medium, where it hydrolyses the ampicillin. After some time Amp concentration in the medium is reduced to a level that allows cells lacking the plasmid to grow too. (In agar plates one can see the presence of "satellites" smaller colonies around the Amp^r colonies which are larger.) In order to avoid this problem: 1. Use the more stable carbenicillin instead of ampicillin. 2. Grow cultures (especially starters) no longer than 8-10 hours.
Tetracyclin is light-sensitive. Grow Tet^r cells covered from light.

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