Question

I'm using Kanamycin for selection after electroporation of my plasmid containing a kanamycin-resistance gene at a concentration of 50mg/L prepared in 1000x stock that is usually kept in frozen aliquots.

When preparing LB plates for selection, I usually prepare a 300ul dilution of the 1000x stock (30ul kan + 250ul h20) and spread that onto my plates evenly. This usually works fine.

However, on some occasions, if I let the plates sit for a week or so, I'll go ahead and reapply more antibiotic just in case.

Is there an amount of kanamycin that is toxic to the E. coli, even if they've been successfully transformed? Are there any side effects I should be aware?

Answer 1

Kanamycin at a concentration of 50 µg/mL is commonly used for selecting Kanamycin-resistant E. coli transformed with plasmids. Adding excessive amounts of the antibiotic can be toxic to even resistant strains. It's important to adhere to recommended levels, monitor transformed cell growth, and check for variables affecting resistance levels.

Step-by-step explanation:

When working with E. coli transformations using plasmids with antibiotic resistance genes as selective markers, such as kanamycin resistance, it is crucial to maintain the appropriate concentration of antibiotic in the selection medium. Typically, the amount of kanamycin needed for effective selection of transformed E. coli strains is 50 mg/L or 50 µg/mL. This concentration is generally non-toxic to cells expressing the kanamycin resistance gene; however, excessive amounts can lead to toxic effects, even in resistant strains. The resistance gene allows the bacteria to inactivate or efflux the antibiotic, but it might be overwhelmed if the concentration is too high, or if the antibiotic is repeatedly added, which could lead to reduced growth rates or even cell death.
In your case, where you reapply kanamycin to the LB plates after a week, it is important to be aware of potential side effects. Over time, antibiotic potency can decrease, especially when plates are not stored properly. While reapplication can help maintain selection pressure, be mindful of not exceeding the initial effective concentration. As long as you adhere to the recommended concentration levels and storage conditions, the reapplication of kanamycin should not be detrimental to the transformed E. coli cells.
It is also valuable to note that transformed cells can have varying levels of resistance depending on the expression levels of the resistance gene. Therefore, maintaining a consistent concentration avoids putting unnecessary stress on the transformed bacteria. Variability in kanamycin sensitivity can be due to factors such as plasmid copy number, promoter strength of the resistance gene, and metabolic state of the cells. If you observe that some colonies are not thriving after reapplication of antibiotic, it might be worth evaluating these factors.