Difference between revisions of "Part:BBa K3634018"

 
 
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<partinfo>BBa_K3634018 short</partinfo>
 
<partinfo>BBa_K3634018 short</partinfo>
  
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The ccdAB toxin-antitoxin (TA) module is a type II TA module where the toxic ccdB protein, poisons the enzyme DNA gyrase, required for negative supercoiling of DNA (Bernard and Couturier, 1992). Through ccdB-gyrase complex formation, DNA cleavage results as well as inhibition of transcription by the formation of RNA polymerase roadblocks. The activity of the unstable ccdA antitoxin separates the ccdB-gyrase complex if present (Vandervelde et al, 2017).
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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.
  
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Expression of ccdA and ccdB within the type II TA module is self-regulated by low specificity and affinity of ccdA for individual binding sites of the regulatory region upstream of both ccdA and ccdB genes. This 113bp ccdAB promoter/operator sequence extends into the first ccdA gene and is suspected to have a total of 8 ccdA operator binding sites (Tam & Kline, 1989). The antitoxin binds the operator DNA sites, with some sites overlapping the promoter, functioning as a repressor of ccdAB transcription. The toxin then functions as a co-repressor or de-repressor depending on the molar T:A ratio (Vandervelde et al., 2017).
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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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At low T:A ratio, the operator is repressed however as the ratio is increased, repression is relieved by preferential formation of a V-shaped non-repressing heterohexamer (ccdB-ccdA-ccdB). Specifically, at the moment the molar ratio of T:A > 1, repression is rapidly lost (Vandervelde et al., 2017). Therefore the ccdA antitoxin has dual functionality: to prevent ccdB-gyrase formation and allow for precise transcriptional control of a desired output gene.  
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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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]