Presently I'm working on a host-vector combination, where the E. coli host is K-host and the vector is p15A ori-based vector with tetracycline as selection marker. The problem I'm facing is the stability of glycerol stocks, which is less than a week. If freshly transformed and expressed cells give me 10mg/L yield of the product, a week-old glycerol stock of the same cells gives me around 4mg/L in identical expression conditions. What could be the reason and how can this be handled?
Thanks in advance.
PS: I need to maintain 0.1-2% glucose in the culture in order to avoid leaky expression.
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