Hi,

did you check if your ampicillin is still fine? Once we had trouble when we stored it at 4°C, it was degraded. Since them we storage it in small aliqouts at -20°C.

I used "new" amp 100 stock (100mg/mL - 1g of ampicillin sodium salt + 10mL milli Q water - filter sterilized). I purchased "new" one in one week before so the ampicillin stock should be correct one.

We use ampicillin working solution (stored in 4°C) for 15 days only as it can degrade and may result in plasmid curing (loss of plasmid and hence expression of gene of interest).

You can make new ampicillin stock from powder and perform the experiments from initial clones (giving good protein).

Thanks,

Ampicillin works by inhibiting the bacterial cell wall synthesis, preventing growth of non-resistant cells. Your plasmid has the ampicillin resistance gene (encoding beta-lactamase), which confers resistance to the cells containing the plasmid, allowing them to keep growing. Thus, if everything works as intended only the cells with the plasmid can keep replicating, ensuring that the plasmid is maintained in the population and forcing the cells to produce your target protein. Unfortunately, ampicillin has several drawbacks as a selection agent:

1) Ampicillin is unstable and prone to degrade on its own.

2) The mechanism of ampicillin resistance involves the actual breakdown of ampicillin, reducing its medium concentration over time.

3) To make matters worse, beta-lactamase is secreted from the producing cells, leading to breakdown of ampicillin in the medium, thus protecting also cells that lose the plasmid.

Your plasmid (pET14b) is a very strong expression system based on the T7 promoter and a high copy number. Thus, expressing proteins from this plasmid puts a heavy burden on the cell, especially for toxic or difficult to produce proteins, which is often seen by arrested cell growth after induction. There is therefore a strong selection in favour of removing the plasmid from the cell population unless there is sufficient antibiotic selection pressure to counter it, which is likely what you see (no product but increased cell mass).

In general, I would recommend using other antibiotics not dependent on beta-lactamase, since this is well-known to be prone to these problems (for instance, if you incubate ampicillin-containing plates for an extended period of time you can observe this in the form of small colonies arising surrounding the main, ampicillin resistant cells, as the antibiotic is being degraded in a circle around them over time). But given that you probably don't want to switch production system, I would suggest that you look into testing carbenicillin, which is a more stable variant of ampicillin and should hopefully work better with your current construct.