Preparing a culture of plasmid-harboring Escherichia coli is the starting point in all plasmid isolation protocols. Suboptimal culture conditions and poor bacterial growth can lead to low yield and loss of the plasmid. The following points must be considered when planning to prepare a culture of E. coli for plasmid isolation.
- Aseptic technique
- Inoculum
- Culture type
- Culture medium
- Selective pressure
- Culture conditions
- Culture volume
- Plasmid size and copy number
Aseptic technique
In general, all microbiological works must be carried out in aseptic conditions to prevent contamination of other bacteria/microorganisms. Contamination with other bacteria can result in no or low yield. The contamination problem becomes more dramatic when the contaminated bacteria contain a similar-size plasmid. Therefore, it is advisable to confirm the identity of plasmid by restriction digestion.
Inoculum
An isolated well-grown colony on the selective LB-Agar plate (with appropriate antibiotic) is the major source of bacterial cells frequently used to inoculate a small amount of antibiotic-containing culture medium (1 – 10 ml). Bacterial colonies on LB-Agar plates can be obtained by either streaking the LB-Agar plate with stored bacterial stock (frozen stock or stab culture) or by plating the freshly transformed competent cells with a desired plasmid.
Some researchers inoculate LB medium with bacterial cells directly taken from stored frozen bacterial stock. This is considered a bad practice and can result in a low yield or loss of plasmid. Therefore, it is highly recommended to streak the stored bacterial stock onto the LB-Agar plate and then use a single colony to inoculate the culture medium. Streaking bacterial cells onto an LB-agar plate from the bacterial stock also enables us to analyze colony morphology, size, and growth characteristics which is helpful to identify possible contamination.
Culture Type
Generally, a liquid culture is prepared by inoculating a culture medium containing an antibiotic with a single colony picked from a selective LB-agar plate. In some situations, a well-grown colony can also be taken to isolate plasmid DNA. However, due to the limited number of cells in a colony, the plasmid yield may not be as high as that obtained from a regular miniprep. An alternative approach is to restreak the colony onto an LB-agar plate in an area of 1 cm, which provides sufficient cells for a miniprep. This method is especially powerful when you don’t have sufficient resources (e.g., limited space in the shaker incubator) to prepare a large number of small cultures for screening of colonies in molecular cloning experiments.
Culture medium
The culture medium must provide sufficient nutrients to support the rapid growth of E. coli. A rich culture medium that ensures rapid bacterial growth is typically used. LB is the most commonly used growth medium for laboratory strains of E. coli, as most plasmid isolation protocols and commercially available kits are standardized for this medium. The recommended initial volumes of E. coli culture and buffers for lysis and downstream processes in these protocols and kits are suitable for the cell density achieved by the overnight culture of E. coli in the LB medium. However, several highly rich growth media, such as Terrific Broth, are also available and can be used to culture E. coli. These culture media support much higher saturation densities, which can help reduce the initial E. coli culture volume.
Selective pressure
Plasmid is an extra burden on E. coli cells as cells spend energy and resources to replicate and maintain it. Bacterial cells that lose the plasmid become the dominating population in culture if there is no selective pressure as they have a growth advantage over plasmid-containing cells. Therefore, Plasmid must provide a selective advantage to cells that maintain it.
All plasmid vectors carry at least one antibiotic-resistance gene, which enables plasmid-harboring bacteria to survive and divide in the presence of a respective antibiotic, thus providing them with a selective advantage that forces bacterial cells to maintain plasmid. Remember that the antibiotic must be present at all stages of bacterial culture including when cells are streaked onto an LB-agar plate (selective LB-Agar plate) or inoculated in liquid LB broth. Another important point to consider is antibiotic concentration. Normally, a higher concentration of antibiotic is used for a high copy number of plasmids.
In the absence of the antibiotic, bacteria will lose the plasmid, which will result in low or no yield. The stability of antibiotics is an important factor in culture conditions. Antibiotics are vulnerable to degradation. Antibiotic resistance genes can also act by degrading/inactivating the antibiotics. Satellite colonies appearing on the ampicillin LB-Agar plate are an example of the degradation of ampicillin in culture conditions. Ampicillin resistance gene product (β-lactamase) is secreted out from the bacterial cells which degrade ampicillin in the surrounding areas, that result in the growth of bacterial cells that otherwise would stay quiescent in the presence of ampicillin.
Culture conditions and incubation time
Once the culture medium is inoculated, it must be grown in a shaker incubator at 37°C. Culture conditions should allow the bacterial cells to grow rapidly and culture should follow the characteristic growth curve. Proper aeration is an essential requirement for the rapid growth of E. coli culture. To make sure proper aeration, one must consider the following points:
- Use a culture vessel that has a volume at least 4 times the volume of the culture medium.
- Maintain the shaking speed between 200 – 300 rpm to allow vigorous shaking
- Loosen the culture vessel cap to allow air exchange
The culture is incubated with vigorous shaking for 12 -16 h (overnight). This corresponds to the transition from the log phase into the stationary phase. This phase is characterized by a high plasmid-to-RNA ratio. Longer incubation can cause cell death and contamination of genomic DNA in isolated plasmid DNA.
Culture volume and plasmid yield
The easiest solution to obtain a sufficient amount of plasmid is to prepare the right amount of culture. Depending on how much plasmid is required, culture volume is taken. Small-scale plasmid isolation requires a small volume of culture (1 – 3 ml) and is sufficient for screening of colonies by restriction digestion or by PCR. However, for storage, distribution, and experiments, a large amount of plasmid is prepared. Depending on how much volume of culture is taken for plasmid isolation, the procedures are called miniprep, midiprep, and maxiprep. Miniprep requires a small amount of culture, while midiprep and maxiprep require medium and large volumes of culture respectively.
A small volume of culture can easily be prepared by inoculating a small volume of culture medium (1 – 10 ml) with a single colony and growing it overnight in optimum culture conditions. However, exceeding the culture volume above 10 ml requires the preparation of a starter culture that is used for inoculating a large volume of culture medium. A starter culture can easily be prepared by inoculating a small volume of culture medium (1 – 10 ml) with a single bacterial colony and is grown to reach the culture’s exponential growth phase which takes generally 8 – 10 h. This starter culture is used to inoculate large culture volume, generally at a ratio of 1: 500 or 1: 1000.
Plasmid size and copy number
Plasmid size and copy number have an impact on the yield of plasmid DNA at the end of the isolation process. Generally high copy number plasmid results in good yield as compared to a low copy number plasmid with the same culture volume. Most currently used plasmid vectors are high-copy number plasmids. However, when a significantly large DNA fragment is cloned in them, their copy number is reduced per cell and the expected yield is also less.
