Antibiotics are important tools used to select for particular cell populations. For example, when bacteria are transformed with a plasmid, antibiotics are used to kill or stall the growth of any individual cells that don’t possess that plasmid.

Bacteria – both natural species and lab strains – can harbor more than one plasmid at a time. In the lab, multiple antibiotics are used to ensure that growing cells contain each plasmid that’s being used.

Multiple antibiotics are used to maintain multiple plasmids in bacterial culture. Additional antibiotics slow culture growth and can impact potency. Yet, a few antibiotic pairs are routinely used together, and there are additional strategies that minimize the number of antibiotics needed.

In this article, we’ll discuss the considerations for using multiple antibiotics in bacterial cell culture, and a workaround for using a single antibiotic in these situations.

 

Article Table of Contents

When are multiple antibiotics used in bacterial cultures?

Considerations when combining antibiotics in bacterial cultures

Combining antibiotics changes their potency

Side effects from antibiotics

Alternatives to using multiple antibiotics

References

 

 

When are multiple antibiotics used in bacterial cultures?

One of the most common uses for multiple antibiotics in bacterial culture is for expressing tricky proteins or multiple proteins as a complex. In both of these cases, two antibiotics are needed to maintain selective pressure to make sure each cell contains both plasmids.

Let’s start with when two proteins are being co-expressed from separate plasmids. This is sometimes done when a single protein doesn’t express well on its own. Maybe the protein goes into inclusion bodies, making it difficult to be recovered.

Often, such proteins can be successfully expressed when exposed to an interacting partner. So, by expressing both proteins together, you can rescue the insolubility or otherwise poor behavior of the first protein (Figure 1).

Figure 1. Left, insoluble individual proteins (purple ovals) expressed from the plasmids (gray ovals with purple rectangle) form inclusion bodies at the edges of the bacterial cells. Right, when expressed together with its partner protein (orange rectangle) the resulting protein complex is soluble.

 

Using this strategy is not just a last-ditch effort if a protein has bad behavior. If you’re interested in studying a protein complex together, expressing and purifying both proteins together can save time and effort compared to expressing each individual component by itself then adding them together at the end.

Let’s move on to tricky proteins. I’m intentionally using this phrase as a general “catch-all” term. In this case, the second plasmid isn’t a protein that is going to be co-purified with the first protein. Rather, the second plasmid is a helper that will assist the first protein be better expressed or behaved.

One example of this includes a plasmid that expresses tRNAs that are common in human genes but rare in Escherichia coli genes (Tegel et al, 2010). Another example is a plasmid that expresses a molecular chaperone to help the protein fold correctly (Figure 2) (Hoffmann & Rinas, 2004).

Figure 2. Second plasmids can supplement rare tRNAs (left) or express a molecular chaperone (MC) to help the protein of interest fold (right). When expressing two plasmids two antibiotics are needed to select for each resistance gene (RG1 and RG2).

 

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Considerations when combining antibiotics in bacterial cultures

There are two main issues you should be thinking about when combining antibiotics in cell culture. While both of these are important considerations in mammalian cell culture, usually side effects from antibiotics are less of an issue during the relatively short duration of bacterial cultures.

·         Combining antibiotics changes their potency

·         Side effects from antibiotics

 

Combining antibiotics changes their potency

Synergy is when one antibiotic is more potent in the presence of another antibiotic. Antagonism is the opposite. In this case, an antibiotic works worse with another antibiotic compared to by itself. Most antibiotic pairings are additive, meaning they combine as you would expect without being synergistic or antagonistic.

Unfortunately, whether antibiotics are synergistic, antagonistic, or additive varies depending on which type of cell they’re being used with (Brochado et al, 2019).

Table 1 lists common antibiotic pairings for E. coli. However, if you’re using a different pairing of antibiotics or an uncommon type of cell, then it is worth testing your antibiotics to see if they’re synergistic or antagonistic. 

 

Table 1. Common antibiotic combinations for dual selection in E. coli.

Antibiotic 1	Antibiotic 2
Ampicillin	Kanamycin
Ampicillin	Chloramphenicol
Kanamycin	Chloramphenicol

 

 

Side effects from antibiotics

The intended effect of antibiotics is to kill or stall the growth of contaminating or untransformed cells. However, antibiotics can also change transformed cells in other, more subtle ways, such as perturbing their gene expression, metabolism, or differentiation (Nygaard et al, 2015; Ryu et al, 2017; Varghese et al, 2017).

These side effects tend to be more of an issue in mammalian cell culture for two reasons. The first is that the selection protocols are longer because mammalian cells grow slower. So, mammalian cells are exposed to antibiotics for longer periods of time.

The other reason is that researchers are often trying to study the same properties in mammalian cells that are perturbed by antibiotics.

If you’re studying the metabolism, gene expression, etc. of bacteria, then you will also need to make sure that antibiotics aren’t impacting your read out.

However, in the case of using bacterial cultures for protein expression, you’re really just worried about the proteins being produced and other side effects on the transformed bacterial cells are not critical. The one side effect you’ll likely notice is that selection with multiple antibiotics leads to the bacterial cells growing slower. This is not a big deal, but it will delay inducing protein expression with IPTG by a little bit as you wait for the cells to grow.

 

Alternatives to using multiple antibiotics

It’s possible to transform bacteria with ~ 10 plasmids, with the exact number depending on which species you’re using (Kiattisewee, 2025). However, to stably maintain every plasmid you would need a separate antibiotic for each one, which would lead to some very slow growing cells.

Let’s simplify the problem a little bit since most experiments don’t need 10 plasmids. Imagine you have two plasmids you’re working with. If you don’t want to use multiple antibiotics, you could instead re-clone the gene of interest from each plasmid into one, dual expression plasmid (Figure 3). Then you’ll only need a single antibiotic to select for both genes in the same plasmid.

Figure 3. Two proteins can be expressed from two separate plasmids, left, or from a single plasmid, right. You’ll need an antibiotic (bottom) for each plasmid that corresponds to that plasmid’s resistance gene (RG).

 

GoldBio is your reliable source for high-quality antibiotics, bacterial competent cells, IPTG, and more. Check out the product links below, and throughout the article, for more details on our affordable and high-performing research reagents.

 

 

References

Brochado, A. R., Telzerow, A., Bobonis, J., Banzhaf, M., Mateus, A., Selkrig, J., Huth, E., Bassler, S., Zamarreño Beas, J., Zietek, M., Ng, N., Foerster, S., Ezraty, B., Py, B., Barras, F., Savitski, M. M., Bork, P., Göttig, S., & Typas, A. (2018). Species-specific activity of antibacterial drug combinations. Nature, 559(7713), 259–263. https://doi.org/10.1038/s41586-018-0278-9

Hoffmann, F., Rinas, U. Roles of Heat-Shock Chaperones in the Production of Recombinant Proteins in Escherichia coli . In: Physiological Stress Responses in Bioprocesses. Advances in Biochemical Engineering, vol 89. Springer, Berlin, Heidelberg. https://doi.org/10.1007/b93996

Kiattisewee C. (2025). How many plasmids can bacteria carry? A synthetic biology perspective. Open biology, 15(7), 240378. https://doi.org/10.1098/rsob.240378

Nygaard, U. H., Niehues, H., Rikken, G., Rodijk-Olthuis, D., Schalkwijk, J., & van den Bogaard, E. H. (2015). Antibiotics in cell culture: friend or foe? Suppression of keratinocyte growth and differentiation in monolayer cultures and 3D skin models. Experimental dermatology, 24(12), 964–965. https://doi.org/10.1111/exd.12834

Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., & Ahituv, N. (2017). Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Scientific reports, 7(1), 7533. https://doi.org/10.1038/s41598-017-07757-w

Tegel, H., Tourle, S., Ottosson, J., & Persson, A. (2010). Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3). Protein expression and purification, 69(2), 159–167. https://doi.org/10.1016/j.pep.2009.08.017

Varghese, D. S., Parween, S., Ardah, M. T., Emerald, B. S., & Ansari, S. A. (2017). Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro. Stem cells international, 2017, 2451927. https://doi.org/10.1155/2017/2451927

 

 

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antibiotics bacteria bacterial cell culture cell culture Simon Currie

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