Difference between revisions of "Part:BBa K3634018"
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| − | ( | + | 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 constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence. |
| + | |||
| + | Reznikoff et al. (1978) mutated the regulatory region in question at three different sites. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein. As a result of these mutations, gene expression mediated by the PL8-UV5 promoter will be independently regulated by intracellular concentrations of the lacI repressor as all CAP-associated regulation has been removed. | ||
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Latest revision as of 11:19, 9 August 2020
PL8-UV5
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 constitutive and can be further activated by the catabolite activator protein (CAP), with binding site upstream of the promoter sequence.
Reznikoff et al. (1978) mutated the regulatory region in question at three different sites. Within the CAP binding site, bases -66 (G) and -55 (C) of the wt binding region were substituted with A and T respectively to prevent binding of the CAP protein at low glucose concentrations. The wt -10 promoter sequence was also mutated from TATGTT to TATAAT in order to allow σ factor (RpoD) to bind without relying on further activation by the CAP protein. As a result of these mutations, gene expression mediated by the PL8-UV5 promoter will be independently regulated by intracellular concentrations of the lacI repressor as all CAP-associated regulation has been removed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]