PlacIq + LacI Repressor (+ mf-Lon deg. tag)
The lac operon found in E.coli consists of the three lactose metabolising genes lacZ, lacY and lacA which when expressed, allow the bacteria to use the sugar as a source of energy. The initial regulatory mechanisms in the pathway were outlined by Jacob and Monod in 1961, where the topic of inducible and repressible enzyme systems was discussed. In this system, the transcriptional repressor is a protein known as Lac I which binds to DNA at various operator sequences (termed O1, O2 and O3) which exist both upstream and downstream of the transcriptional start site (TSS). Interaction between the Lac I and operator sequences reduces transcription of the downstream lactose metabolising genes unless relieved by the lactose isomer allolactose. In the absence of Lac I, transcription is weakly constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence (Oehler et al., 1990).
BBa_K3634015 is a fusion between the lacI gene and the mf-Lon degradation tag, a specific sequence recognised by the mf-Lon protease which allows for rapid endogenous breakdown of the protein to which it is attached. The degradation tag used here is the strongest characterised sequence for the specific protease (BBa_K2333001) and will allow for efficient removal of Lac I from the cell when mf-Lon protease is expressed (BBa_K3634014).
A modified version of the lacI promoter (lacIq - featuring an 'up' mutation 20bp into the sequence (-35 region: c -> t)) is also present in the composite part, allowing RNA polymerase to bind the promoter more tightly than the original which leads to a ten-fold increase in lacI expression and therefore a reduction in leaky expression from lacP (Calos, 1978)(Glascock & Weickert, 1998).
Sequence and Features
- 10COMPATIBLE WITH RFC
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- 1000COMPATIBLE WITH RFC