Plasmids need antibiotic resistance genes so that you can specifically isolate bacteria containing your constructs of interest. But does it matter which antibiotic resistance gene you select? In this blog, we’ll break down the mechanism of action of the most common antibiotic resistance genes and which applications are best (or worst) for each antibiotic.
The use of antibiotic resistance
DNA plasmids are traditionally grown in bacteria as a way to produce more of the plasmid, isolate individual plasmids, or express a gene contained in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive a copy of the plasmid or will lose it during division. These non-plasmid-containing bacteria can compete with and confound the goal of your experiment — to grow, express, or isolate plasmids. The presence of an antibiotic resistance gene is a mechanism to culture only bacteria which have retained a functional copy of your plasmid of interest, preventing this issue.
The genes and their antibiotics
Ampicillin, often just called amp, is the historical king of the antibiotic resistance realm. Amp is a semisynthetic derivative of the more widely known antibiotic, penicillin, and is a beta-lactam that inhibits cell wall synthesis. The AmpR (amp resistance) gene produces beta-lactamase to degrade amp and therefore prevent cell death. One of the cool things about amp is that it's exclusively toxic to dividing cells. The other antibiotics listed below affect continuous cellular processes (e.g., protein synthesis), while amp only affects the cell wall synthesis step during cell division. This is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase prior to division, instead of immediately experiencing the effects of the antibiotic. This buys you a little more time, since bacteria won’t divide immediately after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period of 30 minutes is possible with AmpR.
So why even look at another antibiotic if you’ve got amp? Well, amp is less stable than its counterparts and is also known to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted. Satellite colonies are small colonies of bacteria which do not contain the plasmid and its associated resistance gene. They can grow proximal to a colony that does contain the plasmid that essentially ‘eats up’ the amp around it by producing a lot of beta-lactamase.
Pros: Widely available, cost-effective, timesaving for transformations.
Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic environments.
Carbenicillin, colloquially known as carb, belongs to the penicillin family of antibiotics. It interferes with cell wall synthesis as its mode of action (sound familiar?). Carb is degraded by beta-lactamase, just like amp is. Thus, the AmpR gene confers resistance to both carb and amp, due to their common degradation mechanism.
How to choose between the two? Carb is generally a ‘better’ antibiotic than amp: it is more stable in high pH environments and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming while effectively selecting plasmid-containing bacteria.
So… why do some people still use amp? Carb is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately, amp can easily be replaced with carb if satellite colonies are an issue.
Pros: Very stable, interchangeable with Amp, timesaving for transformations.
Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance gene, NPTII, facilitates phosphorylation of kan, which inactivates it. The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for eukaryotic antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection, whereas other plasmids must carry both a bacterial and a mammalian antibiotic selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes).
Pros: Widely available, cost-effective, confers mammalian resistance to G418.
Con: Slow transformation
Spectinomycin and streptomycin both inhibit protein synthesis as their mode of action and have a very similar relationship as amp and carb (they are interchangeable). Like carb, spectinomycin is the more stable and expensive antibiotic compared to streptomycin.
Spectinomycin/streptomycin can be successfully used for plant and bacterial culture, but some types of bacteria, such as SHuffle cells, are already resistant to this antibiotic due to stable integration of resistance genes in their genomes. If you need a plasmid that can go between plants and bacteria, it’s a great option! Otherwise, since it isn’t universally compatible with all bacteria used in research, it isn’t a widely popular selection agent.
Pros: Stable (spectinomycin).
Cons: Cost (spectinomycin) or stability (streptomycin), doesn’t work for all bacteria.
Zeocin is a member of the bleomycin family of antibiotics, which induce cell death by intercalating into DNA and generating DNA breaks. The Sh ble gene provides zeocin resistance by expressing a protein that directly binds to zeocin, preventing it from binding DNA. Zeocin is an all-in-one antibiotic — it is usable in eukaryotic cell culture, yeast, bacteria, and even plants!
The zeocin resistance gene is present in bacteria that express the Tn5 element, but thankfully, most competent cells do not express this factor. Zeocin is handy because it can be a one-stop shop for selection across systems and species. The cost? Zeocin is genotoxic when not 100% inhibited, meaning it will induce mutations in the host/plasmid DNA during selection if it’s not completely bound up. While this likely occurs at a low rate, there is evidence that zeocin is never entirely detoxified, even when Sh ble is expressed (Trastoy et al). Thus, it may not be a good choice for experiments in which off-target mutations are undesirable.
Pros: Works across many species/systems.
Cons: Doesn’t work for all bacteria; may cause plasmid/host DNA damage.
Choosing an antibiotic resistance
Choosing the best antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your needs are, the more you may need to pay attention to antibiotic selection. If you feel like multiple of the above antibiotics would work for you, they likely do! Give our summary table a look for a quick recap of the pros and cons of each antibiotic and happy selecting!
|Antibiotic Resistance||Stability||Time Saving?||Works for multiple organisms?||Compatible with most bacteria?||No satellite colonies?|
|Ampicillin||Fair||Just bacteria||Satellite colonies|
|Kanamycin||Excellent||No||Bacteria and eukaryotes (G418)|
|Spectinomycin||Excellent||No||Just bacteria||Not SHuffle cells|
|Zeocin||Excellent||No||Bacteria, plants, and eukaryotes||Not Tn5-containing cells|
References and Resources
Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks, G. F. (1986). Effect of storage temperature and pH on the stability of antimicrobial agents in MIC trays. Journal of Clinical Microbiology, 23(5), 959–961. https://doi.org/10.1128/jcm.23.5.959-961.1986
Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., & Berkmen, M. (2012). SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial Cell Factories, 11, 56. https://doi.org/10.1186/1475-2859-11-56
Trastoy, M. O., Defais, M., & Larminat, F. (2005). Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage/gei016I
Resources on Addgene.org
Resources on the Addgene blog
- Plasmids 101: Antibiotic Resistance
- Plasmids 101: Blue-White Screening
- Choosing Your Perfect Plasmid Backbone
Topics: Plasmids 101
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