Plasmids 101: Antibiotic Resistance Genes

Posted by Marcy Patrick on Jan 30, 2014 10:29:00 AM



AmpcillinResistance to antibiotics is a widely used tool in molecular biology, yet scientists rarely stop to think about how much easier it makes our lives. Plasmid transformation into E. coli is a fairly inefficient process– just 1 out of 10,000 cells on average! Without some means of quickly determining which cells successfully received the correct plasmid, scientists would spend hours to days trying find their correct clones. Additionally, the presence of a plasmid is disadventageous from the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal DNA, costing additional resources to maintain the plasmid. Adding an antibiotic resistance gene to the plasmid solves both problems at once – it allows a scientist to easily detect plasmid-containing bacteria when the cells are grown on selective media, and provides those bacteria with a pressure to keep your plasmid. Viva la (bacterial) resistance! 

What are antibiotics?

Antibiotics are generally defined as agents that kill bacteria, or inhibit their growth. Although originally sourced from natural products, many common antibiotics used in labs today are semi-synthetic or fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill bacteria (bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction between the two categories may be a bit of a gray area as some bacteriostatic reagents can kill bacteria when used at high concentrations (and vice versa). Looking around the lab, you'll likely find many of the antibiotics listed in the table below. Note, in this post we'll focus primarily on antibiotics against Gram negative bacteria. In future posts, we'll detail selection in non-bacterial cells such as yeast or mammalian cells.

 

NameClassMode of Action* Working Concentration**
Kanamycin

aminoglycoside

Binds 30S ribosomal subunit; causes mis-translation Bactericidal 50-100 ug/mL
Spectinomycin aminoglycoside

Binds 30S ribosomal subunit; interrupts protein synthesis

Bactericidal

7.5-50 ug/mL
Streptomycin aminoglycoside

Inhibits initiation of protein synthesis

Bactericidal

25-100 ug/mL
Ampicillin

beta-lactam

Inhibits cell wall synthesis Bactericidal 100-200 ug/mL
Carbenicillin

beta-lactam

Inhibits cell wall synthesis Bactericidal 100 ug/mL
Bleomycin

glycopeptide

Induces DNA breaks Bactericidal 5-100 ug/mL
Erythromycin macrolide

Blocks 50S ribosomal subunit; inhibits aminoacyl translocation

Bacteriostatic

50-100 ug/mL in EtOH
Polymyxin B

polypeptide

Alters outer membrane permeability Bactericidal 10-100 ug/mL
Tetracycline tetracyclin

Binds 30S ribosomal subunit; inhibits protein synthesis (elongation step)

Bacteriostatic

10 ug/mL
Chloramphenicol   Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH

*In prokaryotes.     **Dissolve in dH2O and sterile filter unless otherwise specified.

The above table lists some antibiotics commonly found in the lab, their mechanism for killing bacteria, and general working concentrations. For instructions on how to prepare antibiotic stocks, see Addgene's Reference Page.

How else can antibiotics be used in the lab?

Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scienists introduce an antibiotic resistance cassette within the coding region of the gene they are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation. When designing these types of experiments it is best practice not to use the same resistance cassette for the mutation and for plasmid selection. Additionally, scientists can use the loss of resistance as a marker for successful cloning. In these instances, the cloning vector typically has two separate resistance cassettes and your gene of interest is cloned into/inactivates or completely removes (in the case of Gateway cloning) one cassette. Counterselection allows the scientist to select bacteria that are only resistant to the antibiotic that remains intact.

Tips and tricks from the bench:

  • Use fresh stocks. Most antibiotics are stable in powder form, but quickly breakdown in solution. Storing aliquots at -20oC and avoiding repeated freeze/thaw cycles will keep most antibiotics viable for at least 6 months.

  • Ampicillin breaks down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite colonies!

  • Carbenicillin is more stable than Ampicillin and can be used in place of Ampicillin in most applications.

  • Antibiotics vary in their sensitivitly to heat and/or light – do not add them to media hotter than about 55oC and store plates/stocks wrapped in foil if a light-sensitive antibiotic like Tetracycline is used.

  • Keep in mind that some E. coli strains have natural antibiotic resistances, so make sure your plasmid and E. coli strain are compatible! Check out this list of common E. coli genotypes and their natural resistances.

 Additional Resources on the Addgene Blog

Resources on Addgene.org

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Plasmids 101

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