loss of Ecoli over-expression

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    • #6794
      cbourne
      Participant

      We are expressing a 150 amino acid protein in pET11b vector in BL21 DE3 e coli cells. This over-expression is very leaky in LB media and we typically don’t have to induce with IPTG. This protocol has been successful in this lab for at least 5 years now.

      Suddenly, twice within 1 year we are having troubles that seem to be specific to LB powder stocks. They will work initially, but within a few months the over-expression is completely lost. The cells grow to roughly the same density and pellet weight.
      We’ve tried:
      -different clones
      -different antibiotic bottles/stocks
      -different water
      – plus or minus IPTG induction
      -different LB stocks/ powders – older bottles of LB work fine, but using our fresh powder stocks expression is again lost!

      Anyone have any thoughts- what could be changing to specifically inhibit over-expression (T7 promoter) but not affect e coli growth?

      Thanks for any help,
      Christina

    • #68055
      joveryant
      Participant

      It’s ver surprising!!

      try different temperature & different competent cell

      good luck

    • #68604
      oppox
      Participant

      to inhibit expression u could try tuner or rosetta cells (these are from novagen other companies have different ones) they contain an extra plasmid for expression of the pet-blue which lacks the T7 thingy. Though rosetta is also a rare codon cell strain so maybe tuner is the first to start with.

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