The culture wars

Plasmid vectors are ubiquitous in modern biological research and are produced in quantity by culture and subsequent extraction and purification from the bacterial host E. coli. This post outlines how to properly culture plasmid-hosing E. coli to ensure consistently high yields of plasmid vectors.

Plasmid vectors are an essential, ubiquitous, and powerful tool in modern life science research, yet they can often be a point of difficulty or failure in key experiments. The modern academic and industrial research environment dictates that researchers maximize use of precious time and funding by ensuring efficient creation and use of these basic tools.  Many experimental applications require the reliable and regular use of relatively large quantities of high-quality vector DNA, including sterile and endotoxin-free preparations in cell culture. An essential step in maximizing plasmid yield is proper culture conditions for the plasmid-hosting bacterial cells.

Whether preparing a small amount of vector for analysis or milligram-plus quantities for transfection of mammalian culture cells; application of the methods described here will ensure healthy high-yield cultures of vector-carrying E. coli such as in the title image above, giving you a reliable and abundant supply of high-quality plasmid DNA for your research needs. In our last post we discussed identification and recovery of mislabelled plasmids and dead glycerol stocks. Having a vector in hand, in this post we will deal with the next area of potential difficulty – the culture wars.

Selectable markers and antibiotics

The most important thing to remember when choosing a selectable marker is that you will rarely have a choice in selectable marker. Most often, we choose a vector based on the insert, reporter target, propagation, or expression platforms it was designed for and are obliged to use whatever selectable marker it comes with. The selectable marker forces maintenance of the vector DNA by the host cell in the presence of a selective pressure – in this case an antibiotic added to the growth medium. Therefore, the proper application of antibiotics in vector propagation is an essential step for success.

While a plethora of selectable markers exist for the maintenance of plasmid vectors in E. coli, the NeoR/KanR and AmpR genes are by far the most common. NeoR/KanR encodes an aminoglycoside phosphotransferase originally derived from the Tn5 transposon and confers kanamycin resistance in bacteria and neomycin or G418 resistance in eukaryotes. The AmpR gene encodes the enzyme β-lactamase which confers resistance to β-lactam antibiotics such as ampicillin and carbenicillin by hydrolyzing and inactivating them. AmpR was originally obtained in 1963 from a naturally occurring plasmid mediating ampicillin resistance in Salmonella paratyphi B and subsequently pressed into service as a selectable marker in plasmid vector propagation in E. coli.

The β-lactamase enzyme is secreted by cells into the medium. This means that ampicillin or carbenicillin are gradually broken down in the medium during culture of resistant cells. This breakdown causes the satellite colonies that may appear on agar plates containing ampicillin and is a cause of gradual plasmid loss from liquid cultures. Kanamycin is not broken down in the media and therefore results in higher rates of plasmid maintenance, particularly at higher culture densities and longer incubation times. For this reason, kanamycin resistance is preferred where simple propagation of vectors is the main goal – such as preparation of vector for subsequent transfection of mammalian cells. However, because kanamycin exerts its antibiotic effect by inhibiting protein synthesis, AmpR vectors are preferred for bacterial protein expression experiments.

Ampicillin is best used at a final concentration of 200 µg/mL, though a better choice is carbenicillin which is more resistant to breakdown by β-lactamase and should be used at 100 µg/mL.  Carbenicillin is more expensive than ampicillin but in our experience, it gives substantially superior results and is what we use exclusively in our liquid and solid media for selection and growth of AmpR vectors. When scaling up cultures of AmpR vectors, such as inoculating a 100 mL liquid culture from 3 mL overnight, it is important to spin down the culture, remove the old medium, and resuspend the cells in the fresh medium. This removes β-lactamase that has accumulated in the culture medium. Kanamycin is used at 50 µg/mL and centrifugation and resuspension are not required when scaling up cultures.

Media

When propagating plasmid vectors in E. coli, lysogeny broth (LB) is often the go-to media. LB media was originally optimized for propagation of bacteriophage in Shigella sp. and although it is not the optimum medium for E. coli in many applications its use remains a persistent tradition. We use LB for our agar plates when propagating E. coli due to its inexpensive composition, but our medium of choice for liquid culture of E. coli is Terrific Broth (TB, 2.4% w/v yeast extract, 2% w/v tryptone, 0.4% w/v glycerol, 0.1 M potassium phosphate pH 7.2). The major benefit of TB is the phosphate buffer which moderates pH of the media during bacterial growth.

In media without buffering agents the pH decreases during culture due to accumulation of acidic metabolic products, leading to a slowing of bacterial proliferation and lower cell yields. Some reports have indicated that media high in phosphate such as TB may interfere with the action of kanamycin as a selection agent, but we have never observed this phenomenon ourselves and routinely obtain high yields of plasmid from cultures of E. coli in TB containing kanamycin.

The influence of media choice on cell and vector yield are shown in Figure 1. In this illustrative experiment we inoculated equal numbers of E. coli transformed with a KanR-vector into 3 mL of either LB or TB containing kanamycin and measured the cell density, total yield of vector DNA, and the yield of DNA per OD unit after 18 h at 37 C. These data show that cell density was nearly four-fold higher in TB than in LB with correspondingly higher total yield of vector. While it is obvious that more cells mean more vector, what about the yield of vector as a function of an equivalent number of cells? The vector yield per OD per mL in Figure 1 shows that a higher yield of vector is obtained from the same number of cells when grown in TB.

Culture conditions

Much is often made of the importance of culture growth phase on plasmid yield. While use of cells in log phase is important for many manipulations in the molecular biology laboratory, it is not critical to maximizing plasmid yield.

The data in Figure 2 show that the per-cell yield of vector is higher during log phase, as previously reported. However, these data also show that the slight difference in per-cell yield is insignificant compared to the much higher overall yield obtained with a stationary phase culture. Nevertheless, cultures should not be allowed to remain for extended periods at stationary phase and the ideal time to harvest cells is at the beginning of stationary phase when viable cell numbers are maximized, about 16 – 18 hours at 37 °C.

In our experience, the single biggest factor affecting vector yield is aeration. E. coli is a facultative anaerobe. Although this means that it can survive in the absence of oxygen, optimum and vigorous growth requires it.

This difference is illustrated in Figure 3, where equivalent numbers of E. coli cells carrying a KanR vector were inoculated into medium that was either purged with nitrogen gas and sealed or incubated with good aeration. Both total vector yield and per OD per mL vector yield were substantially higher when cultures were aerated. 

The best way to ensure aeration of small volume cultures is to ensure you are using culture tubes with loose-fitting caps and shaking the cultures at an angle as shown in Figure 4. Speeds of 160 – 200 rpm on the shaker are generally appropriate and the medium should be well agitated during culture.

For larger volume cultures, the use of baffled flasks as shown in Figure 5 is best.  Keep the volume of liquid small relative to the volume of the flask and ensure the tinfoil or other cover is slightly loose to allow air exchange.  Shaking speed should ensure vigorous mixing of the culture.

Winning the culture wars

If you’ve been suffering from low plasmid yields then remember the key points above:  Use the antibiotics at the concentrations given above, grow your cells in TB media, and ensure good aeration of the culture. With this trifecta of good culture practices, you are sure to consistently obtain high yields of plasmid vector for use in your experiments.

You can also send your vector to Proxima and receive a milligram of endotoxin-free transfection grade plasmid DNA at a price competitive with what it would cost to do it yourself. If you use a lot of vector DNA, consider our plasmid DNA archiving and on-demand production service. Simply send us your vector and for a small annual fee we will maintain it, and when you need some, simply order and receive a milligram of high-quality plasmid in as few as five business days.

Use Proxima’s contact form and stop engaging in culture wars today.