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  • FAQ: Why are there no colonies or no growth in liquid culture (C2529)?

    This outcome is often due to basal expression of a target gene product, which is detrimental to cell viability. Basal expression of target protein in NiCo21(DE3) is identical to basal expression in BL21(DE3). Some vector systems (in particular those utilizing a T7 promoter) allow expression without inducer. If tightly regulated T7 expression is required, use a strain expressing lysY:
    • T7 Express lysY (NEB #C3010) lysY produces a mutant T7 lysozyme which binds T7 RNA polymerase, reducing basal expression of the target protein. Upon induction, newly made T7 RNA polymerase titrates out the lysozyme and results in expression of the target protein.
    • T7 Express lysY/Iq (NEB #C3013) lysY expression as well as lacI over-expression to repress basal expression of the T7 RNA polymerase.
    • Lemo21(DE3) (NEB #C2528) BL21(DE3) containing the Lemo System™. LysY expression is modulated by L-rhamnose, making T7 protein expression tightly regulated and tunable.