I fell in love with biology because of an image that was honestly quite boring. My Bio 101 professor displayed a codon table — a chart every molecular biologist has seen before, showing how DNA sequences are translated into amino acids. And I was astonished that the molecular language shared by every living thing fit onto a single lecture slide.
That shared language is the basis of shuttle vectors, which can function in unrelated host species, even from completely different kingdoms of life. That’s convenient for researchers who want to do experiments in complex eukaryotic cells, but would prefer to grow, clone, and manipulate their vectors in fast-growing bacteria first. In fact, most yeast vectors and many mammalian vectors are designed to shuttle between species.
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| Figure 1: Shuttle vectors are vectors that can function in more than one host species. Created with BioRender.com. |
How are shuttle vectors different from other plasmids?
If we all use the same molecular code, can I transfect any old bacterial plasmid into HeLa cells and get on with my experiment? Not quite. Shuttle vectors have to address several big differences in the ways vectors are used across species: replication, selection, and expression.
Replication
It’s usually not enough to get your vector into your target cells; you also have to consider how to keep it there. In fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the population.
A bacterial chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid needs a compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated bacteria should include oris for both target species.
Fun fact! Plant vectors are usually designed to shuttle between E. coli and Agrobacterium tumefaciens, a plant-infectious bacteria that can incorporate foreign DNA into plant genomes.
Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a partial centromere sequence (CEN), both, or neither, depending on the yeast species and the goal of the experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as a bacterial ori for prokaryotic growth.
Sincere there are no natural mammalian plasmids, mammalian vectors are even less similar to bacterial ones. The mammalian analogue of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is incorporated into the host cell’s genome, so no special sequence is needed to retain the vector as cells divide. In transient transfection, the vector remains episomal, meaning it stays separate from the genome. One option for episomal vectors is to include viral oris to drive amplification, which helps increase cellular uptake, but this approach requires compatible cell lines that express viral machinery.
Alternatively, some slow-growing cells, particularly mammalian cells like neurons, may not replicate during the course of your experiment, meaning you don’t have to worry about vector replication at all. In these cases, transient transfection may be sufficient for your needs even without a compatible replication site.
Regardless of the species you want to be able to shuttle your vector between, you should have a plan to ensure it persists in both contexts.
| For a vector to persist in... | You need... |
| Bacteria |
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| Yeast |
...depending on the yeast species and the goal of the experiment |
| Mammalian cells |
At least one of the following:
|
Selection
Once a plasmid is delivered to your target cells, you still need to select for the cells that contain it.
Antibiotic selection
Most bacterial plasmids used in the lab contain an antibiotic resistance gene to allow for antibiotic selection, but these resistance systems don’t always translate to eukaryotes. For example, beta-lactam antibiotics like penicillin and ampicillin inhibit cell wall synthesis. Eukaryotes, with no cell walls, can grow alongside these antibiotics just fine. This is useful for keeping your eukaryotic cell cultures bacteria-free, but it won’t allow you to select for your vector.
Some antibiotics block important cellular pathways shared by bacteria and eukaryotes. For example, puromycin inhibits protein synthesis across kingdoms by targeting a shared aspect of ribosomal function. A puromycin resistance gene on your vector can protect both plasmid-carrying bacteria and eukaryotes.
But be careful — antibiotic mechanisms vary! Aminoglycoside antibiotics also block protein synthesis, and the neomycin resistance gene protects against any of them. But different aminoglycosides work on different host species: kanamycin targets the prokaryotic ribosome, while G418 (geneticin) is effective at inhibiting the eukaryotic ribosome. One resistance gene on your vector might require two different selection molecules! It all depends on how the antibiotic works.
Another example is Zeocin, a DNA intercalator, which affects all kingdoms of life. Of course, that includes you. Besides the risk to the researcher, Zeocin has been shown to cause mutations even in cells with the resistance gene, so it isn’t a good fit for all experiments.
And the list of antibiotic options doesn’t end there. Make sure you read the instructions for whatever antibiotic you choose to ensure it works in your host species!
Auxotrophic selection
Another option is to use a different method of selection entirely. Auxotrophic selection relies on the host organism being unable to synthesize a nutrient required for growth. Before delivery of the vector, the deficient cells are grown in media that supplies the key nutrient. The vector provides the missing synthesis machinery, and the cells are switched to minimal media, allowing cells that contain the vector to survive while the auxotrophic cells die.
Some auxotrophic selection markers can be used across species, for example, in both S. cerevisiae and E. coli. Auxotrophic selection is especially common in yeast experiments because yeast tend to spontaneously develop resistance to selection toxins.
Auxotrophic selection is useful for producing stable mammalian cell lines, but it isn’t common for simple transfection experiments.
Reporters
Finally, in some cases a researcher may choose not impose selection on cells and instead to use a vector with a reporter, such as a fluorescent protein, to visualize vector uptake. These reporters might be used as markers for automated cell sorting or to visually select cells in a microscopy image for individual analysis. A shuttle vector designed for this approach in a mammalian cell experiment should still include an antibiotic resistance gene for growth in bacteria.
Expression
You’re almost there: you’ve planned for vector replication and encoded a selectable marker for both your target systems. But will your cells actually express your gene of interest? Many promoters are species-specific, so you’ll need to design your vector to be functional in the organism your experiment takes place in.
In many cases, you won’t need to worry about the expression of your gene of interest in both systems you are shuttling between. For example, if you are using E. coli simply for easy cloning and quick DNA production, you probably don’t need it to express your protein of interest. However, you do still need a species-compatible promoter for expressing your selectable marker. Otherwise, you could end up killing bacteria that have taken up an antibiotic resistance gene they simply can’t turn on!
Wait, what even counts as a shuttle vector?
Are viral vectors shuttle vectors?
The production of viral vectors typically involves shuttle vectors, also sometimes called transfer vectors. The gene of interest is cloned into a shuttle vector for transfection into producer cells, which have the machinery to manufacture infectious viral particles. Those particles are collected to deliver the gene to target cells.
However, while shuttle vectors are involved in the production of viral vectors, we consider the viral vectors themselves to be a distinct gene delivery method. We focused this post on shuttle vectors delivered by transformation and transfection and left the business of transduction to our Viral Vectors 101 series.
Are broad host range plasmids shuttle vectors?
While it’s technically accurate to say that shuttle vectors have a “broad host range,” these terms are typically used in different (though overlapping) ways. Broad host range plasmids are those that can replicate in multiple kinds of bacteria, including many natural plasmids. By contrast, shuttle vectors are lab tools, and they include both vectors for unrelated bacterial species and those that can shuttle between prokaryotes and eukaryotes.
Conclusion
I still think it’s pretty incredible that my cells “speak” the same molecular language as the tree outside my window! Whatever you use them for, shuttle vectors can help you hop, skip, and jump between all kingdoms of life. A bit of careful design will help make your experiment with shuttle vectors a success!
Resources
Resources on the Addgene blog
- Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection
- Plasmids 101: Choosing an Antibiotic Resistance Gene
- Plasmids 101: Yeast Vectors
- Plasmids 101: Mammalian Vectors
- Plasmids 101: The Promoter Region
Resources on Addgene.org
Topics: Plasmids 101
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