Enhance Your Culture: How to Improve Bacterial Growth for Efficient Gene Delivery
Cloning serves as the foundation for most gene delivery experiments and is often where initial challenges emerge. After meticulously constructing a vector with the ideal GC content, devoid of secondary structures, and equipped with all the necessary components for experiment success, it can be frustrating when bacterial growth efficiency falls short. At VectorBuilder, we are dedicated to enhancing every facet of your gene delivery experiments and providing invaluable troubleshooting support of your cultures.
Plasmid transformation and growth
To deliver genetic material into your target cells, the initial step involves propagating your plasmid through transformation. Most standard protocols employ LB plates with antibiotics to select for cells successfully transformed with plasmids carrying antibiotic resistance genes. The antibiotic concentration varies depending on the specific antibiotic (Table 1); for instance, ampicillin is recommended at a concentration of 100 µg/mL (50 ug/mL for plasmids with low-copy origin of replication). Another popular screening method involves lacZ. In this approach, the presence of the lacZ gene on your plasmid allows for blue/white screening using a beta-gal derivative. Ensuring the functionality of all selection agents and using appropriate concentrations is pivotal for successful selection. Moreover, it's crucial to plate the right number of cells, as excessive cells can lead to the growth of untransformed cells, while plating an insufficient amount or using an excessively high antibiotic concentration can hinder growth.
|Antibiotic||Recommended concentration||Antibiotic||Recommended concentration|
|Ampicillin||100 ug/mL||Tetracycline||5 ug/mL|
|Kanamycin||50 ug/mL||Streptomycin||50 ug/mL|
|Chloramphenicol||34 ug/mL||Gentamycin||10 ug/mL|
Following transformation, numerous options are available for setting up the growth of your bacterial cultures, ranging from the choice of glassware to the selection of media and incubation times. At VectorBuilder, we've conducted exhaustive testing across various conditions using VB UltraStableTM cells transformed with a plasmid carrying ampicillin resistance.
Cells are commonly incubated in SOC media to facilitate recovery after transformation (Figure 1), but it is not optimal for outgrowth purposes. Our findings show that the plasmid yield from bacteria grown in this medium is much lower than that from bacteria grown in LB. Furthermore, our in-house testing indicated that varying the antibiotic concentration (100-150 µg/mL) in the culture media has negligible impact on plasmid yield. However, it is important to prepare fresh culture media, as antibiotics, ampicillin in particular, degrade quickly in liquid culture. Using stale media may result in the growth of bacteria that do not contain your plasmid.
Figure 1. Typical culture process. Bacteria are incubated in SOC medium for recovery, then plated. An individual colony is then selected and grown in 1-5 mL culture before being transferred to a large flask, if needed.
When setting up cultures, it is important to consider the quantity of plasmid needed, whether you are using plasmids with a high-copy or low-copy origin of replication, and the binding capacity of your prep column. Mini preps produce lower quantities of plasmid because only 1-5 mL of culture is grown overnight, yielding up to 25 ug of high-copy plasmid DNA and 12.5 ug of low-copy plasmid DNA. If a higher quantity of plasmid DNA is needed, midi or maxi prep should be used. For maxi preps of high-copy plasmid, we recommend using 100-150 mL of overnight bacterial culture, which can yield up to 500 ug of plasmid DNA; for low-copy plasmid, we recommend using 300-500 mL of overnight bacterial culture, which can yield up to 200 ug of plasmid DNA. It is important to note that bacterial cultures should not be left to saturate for too long before harvesting, as this can lead to not only antibiotic degradation, but also death of bacterial cells and degradation of plasmid DNA. If immediate extraction is not possible, pelleted bacterial cells can be stored at -80°C for later plasmid prep.
Once cells are moved into larger-scale cultures (e.g. 12 or 500 mL), optimizations can be made to increase final yield. There are many options for culture media, with further modifications possible. From your base media, altering nutrient and sugar sources are a very good starting point. We have found an average 57% increase in plasmid yield using our proprietary medium modified from LB (Figure 2).
Figure 2. Comparison of our modified LB to standard LB for 12 mL and 500 mL cultures using 15 and 55 different plasmids, respectively. Average yield for LB for each culture volume is normalized to 1. Using modified LB produced a significantly higher yield for 500 mL cultures.
While optimizations can be made as general practice, particular plasmids may require individual modifications. In 55 batches of 500 mL bacterial cultures carrying different plasmids, 50 showed increased yields in our modified LB compared to conventional LB, 4 showed lower yields in modified LB, and 1 showed no difference (Figure 3). Additionally, within equivalent culture conditions, we found more than 10-fold variation between batches containing different plasmids. We have discussed how plasmids containing toxic genes can lead to decreased viral titer, and this can also lead to decreased plasmid yield. Additionally, plasmid characteristics including origin of replication, plasmid size, GC content, and secondary structures can impact plasmid yield.
Figure 3. Plasmid yield of 500 mL cultures grown in modified LB relative to LB (dotted line). Each batch represents bacteria containing a different plasmid.
Reviving bacterial glycerol stocks
Glycerol stocks offer an excellent solution for the stable long-term storage and shipment of bacteria. Stocks stored at -80°C can be thawed on ice, and a small sample can be used to inoculate a 2-5 mL LB culture containing the appropriate antibiotic. Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. The colony can then be selected and cultivated, followed by RE digestion and Sanger sequencing to confirm the identity of the plasmid.
As a final recourse, if none of these options produce a viable yield, plasmid DNA can be extracted from the glycerol stock and retransformed into new competent cells (download user instruction). However, this option typically results in a lower plasmid yield and inefficient transformation, underscoring the importance of having appropriate negative controls. Following transformation, cultures can be re-established, and new glycerol stocks can be produced.
Why it matters
The production of a substantial plasmid stock for your gene delivery experiment is a critical endeavor. Whether you want to directly transfect your target cells or proceed to generate recombinant viruses, having an appropriate amount of plasmid DNA is imperative. Troubleshooting your lack of growth allows you to continue your experiment, and optimizing culture media, incubation times, and even the choice of glassware contributes to efficient resource utilization. At VectorBuilder, we're committed to assisting you with every step, big or small, from your complex cloning project to increasing your plasmid yield.
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