Selective Media Used in Gene Cloning

When utilizing antibiotic-based selective media in your cloning experiments, its essential to be cautious and mindful of a few considerations like phenotypic lag and how to differentiate between recombinant and non-recombinant vectors.

Phenotypic lag

Phenotypic lag is the time delay between gene acquisition (from the vector) and witnessing its phenotypic expression.

After vector uptake, cells require 45-60 minutes before expressing the antibiotic resistance gene in the vector and then expressing that phenotype.

Keeping phenotypic lag in mind, culture the host cells in media without antibiotic pressure for 45-60 minutes after transformation. Then you can plate them on antibiotic selective media.

If you culture the recipient cells right after transformation, even the transformed cells that took up the vector might die.

Distinguishing between recombinant and non-recombinant vectors

Antibiotic-based selection tells you which host cells have the vector. But it can’t tell you if a colony has the recombinant vector – that is, the vector with the cloned transgene, or whether it has the non-recombinant empty vector.

Take a look at figure 2 for more clarity.

Figure 2. Shows what happens on an antibiotic selection plate.

In figure 2, you can see that the cells with just the insert (1) and the cells with the linearized plasmid (2) will not survive antibiotic selection pressure.

Cells with the recircularized vector (3), containing genes for antibiotic resistance, and cells with the correct plasmid (4) will survive the selection pressure on the plate.

For the genes on a plasmid to get expressed, the plasmid needs to be in its circular form within the cell. Genes on a linearized plasmid won’t get expressed, and ultimately, a linearized plasmid gets lost in the cell, because it does not get replicated.

While the recipients that have just the insert or the linearized vector won’t survive antibiotic selection, host cells with either empty vector or the recombinant vector will produce colonies on the antibiotic plate.

To figure out which colonies have your desired recombinant vector, you would need to do further analysis – for example, by colony PCR.

Further, when you want to clone multiple inserts together, then antibiotic selection wouldn’t be able to tell you which colony has your desired clone. You would need to confirm by colony PCR, or restriction enzyme mapping of the plasmid construct.

Drop-out media for selection

Drop-out media is used to select host cells that have the vector. This medium lacks a certain nutrient, such as an amino acid or nucleotide, necessary for host survival.

Similar to antibiotic selection, this strategy would not be able to differentiate between host colonies that have the correct recombinant vector versus those with the empty vector.

The recipient host cells are transformed with a plasmid (or any other suitable vector) that has a gene cassette encoding enzymes for synthesizing metabolite 'B,' essential for growth and survival of this organism. This recipient host strain is an auxotrophic mutant for compound B. Following transformation, when plated on media that lacks compound B (that is, this medium is a drop-out for compound B), only the cells that have taken up the vector grow and produce colonies. Cells that have not taken up the plasmid do not survive on this selective medium.

Figure 3. Illustrates drop-out media-based selection of host cells during gene cloning.

Let’s take what we learned and summarize it but with figure 3 in mind. Looking at figure 3, you can see that you would transform the recipient host strain with a vector. This vector has the genes to synthesize metabolite ‘B,’ which is needed by the organism for growth.

Following transformation, you plate the recipient strain cells on a medium that is deficient, or a drop-out for nutrient ‘B’. In other words, the medium that you plate your cells on does not have nutrient B.

The cells that have taken up the vector will survive on this media since they are equipped to produce the absent nutrient, while the cells without the vector won’t survive because they have no means of producing the nutrient essential for their survival.

The primary requirement for selection using drop-out media is that the recipient host strain you are using is an auxotroph for that metabolite.

Auxotrophy is a phenotype of an organism where it cannot synthesize a particular compound/ metabolite required for its growth and is an auxotroph for that compound.

An auxotroph is dependent on the medium supplying the corresponding compound. If the particular culture medium lacks that compound, the auxotroph cannot survive there.

To put things in perspective, the recipient host strain shown in figure 3 is an auxotrophic mutant for compound B.

Experimental considerations for choosing antibiotic versus drop-out media for selection of transformants

Organisms need to be auxotrophic for a certain nutrient for drop-out media selection to work. Likewise, antibiotic selection is going to be more appropriate for certain situations. Here are some key considerations to help you narrow down your choice between these methods.

When you might prefer drop-out media for selection of transformants?

Confirm that your recipient host strain exhibits auxotrophy for a specific compound. The corresponding vector must encode enzymes for synthesis of the corresponding compound. If you have an auxotrophic mutant strain as your recipient host, and a suitable vector that can complement this mutation, this makes it an ideal situation for drop-out media-based selection.

Why some experiments avoid antibiotic-based selection of transformants?

• If artificially introducing resistance to a particular antibiotic in a strain interferes with the downstream assay. Suppose you are studying an organism for its inherent resistance to antibiotic ‘A.’ In this case, you definitely would not want to transform it with a plasmid that encodes resistance to the same antibiotic.
• If the recipient strain is already resistant to the antibiotic you were planning to select with.
• Be very careful if you have artificially made a lab strain resistant to a particular antibiotic using a plasmid. Antibiotic resistant bugs are a leading cause of mortality and morbidity. Though extremely rare, there have been cases where lab strains with plasmids that encode antibiotic resistance escaped the lab. And this can cause a public health concern.
• Organisms such as yeast, widely used in cloning, are inherently resistant to many common antibiotics. They also spontaneously may become resistant to a lot of antibiotics during the course of the experiment. So, when using yeast as the host organism, drop-out media might be a better mode for selecting transformants.

When using these screening techniques in your gene cloning procedure, you are doing positive selection. You are selecting host cells based on the fact that they have a gene on the vector, such as an antibiotic resistance cassette or a gene for a synthetic metabolite.

But, you can also do negative selection to identify your desired host cell colonies. Here you select based on the criterion that the cells do not have a specific gene on the vector. This strategy is common in gene cloning techniques that use homologous recombination.

References

Green and Sambrook. 2012. Molecular cloning-a laboratory manual. Cold spring harbor laboratory press. 4^th edition

Pronk. 2002. Auxotrophic Yeast Strains in Fundamental and Applied Research. Appl Environ Microbiol. 68(5): 2095–2100