Difference between revisions of "Part:BBa K3512012"
 
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<partinfo>BBa_K3512012 short</partinfo>
 
<partinfo>BBa_K3512012 short</partinfo>
  
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The CcdB toxin is part of the CcdA-CcdB system. The target of CcdB is the GyrA subunit of DNA gyrase, an essential type II topoisomerase in Escherichia coli. Gyrase alters DNA topology by effecting a transient double-strand break in the DNA backbone, passing the double helix through the gate and resealing the gaps. The CcdB toxin acts by trapping DNA gyrase in a cleaved complex with the gyrase A subunit covalently closed to the cleaved DNA, causing DNA breakage and cell death in a way closely related to quinolones antibiotics.
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A biosensor is a module that combines biological molecules as the recognition element with a physical transducer or repressor and outputs quantitative data corresponding to the biomolecule’s concentration (Park, M et al., 2013). Generally, these specific interactions between the ligand and the transducer are used to determine secretion or production rates in the form of an electrochemical and/or optical signal. In our case, we coupled the biosensor to the expression of anti-invertase.
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<h2> pFruB - Cra (FruR) Regulation </h2>
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The enterobacterial catabolite repressor/activator (Cra) protein or the FruR protein is a pleiotropic regulator that controls expression of a large number of metabolic genes in response to the flux of glycolytic intermediates in various natural biological systems. The regulator is able to interact with specifically fructose-1-phosphate and regulate the action of pFruB promoter (which is a common promoter usually found in the fructose operon) (Pereira, L.F.M. et al., 2016) (Chavarría, M et al., 2014).
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<h2> Mechanism </h2>
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The FruR expression is under an IPTG-induced pLac promoter (BBa_K3156000) which is induced before packaging the bacteria in our polymer inoculant. FruR is a transcription factor with an affinity for fructose-1-phosphate which is produced from the D-fructose formed after invertase action. The FruR gene (BBa_K2448009) encoding for the FruR protein prevents the transcription of the regulated promoters. pFruB (BBa_K2448017) is the promoter region following FruR and is repressed by the FruR transcription factor, in the absence of D-Fructose. This prevents any further transcription, and no anti-invertase protein is produced. If D-Fructose is present in the cell, the FruR transcription factor will bind preferentially to it and thus be inactivated. This means that the repression of the related promoter pFruB will be released, enabling the transcription of the anti-invertase protein which can then act on invertase and prevent the inversion of sucrose.
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[[Image:Mechanism1.png|450px|thumb|center|]]
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<h2>Mathematical Model</h2>
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<h3>Parameters</h3>
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[[Image:Parameters1.png|450px|thumb|center|]]
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<h3>Differential Equations</h3>
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[[Image:ODE1.png|200px|thumb|center|]]
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Here,
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[P] is the pFruB concentration;
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[R] is the FruR concentration;
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[M] is the mCherry concentration;
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[A] is the anti-invertase concentration;
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[C1] is the concentration of the [F1P-FruR] complex;
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[C2] is the concentration of the [pFruB-FruR] complex (in the binding domain);
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[Rb] is the concentration of the domain-bound pFruB and FruR complex (Bisswanger, H., 2008); and
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[CB]=CNV is the approximate concentration of binding domains per cell where CN is the plasmid copy number and V is the cell volume.
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In absence of the antitoxin CcdA, the CcdB toxin traps DNA-gyrase cleavable complexes, inducing breaks into DNA and cell death.
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3512012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3512012 SequenceAndFeatures</partinfo>
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<h2> References</h2>
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<ul>
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<li>Park, M., Tsai, S.-L., & Chen, W. (2013), Microbial Biosensors: Engineered Microorganisms as the Sensing Machinery. Sensors 13(5), 5777-5795.</li>
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<li>Pereira, L. F. M., Ferreira, V. M., Oliveira, N. G., Sarmento, P. L. V. S., Endres, L. & Teodoro, I. (2017). Sugars levels of four sugarcane genotypes in different stem portions during the maturation phase. Annals of the Brazilian Academy of Sciences. 89(2), 1231-1242.</li>
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<li>Chavarr a, M., Durante-Rodr guez, G., Krell, T., Santiago, C., Brezovsky, J., Damborsky, J., & de Lorenzo, V. (2014). Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator ofPseudomonas putida. FEBS Open Bio 4(1), 377-386.</li>
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</ul>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 18:35, 24 October 2020

pFruB-Cra System

A biosensor is a module that combines biological molecules as the recognition element with a physical transducer or repressor and outputs quantitative data corresponding to the biomolecule’s concentration (Park, M et al., 2013). Generally, these specific interactions between the ligand and the transducer are used to determine secretion or production rates in the form of an electrochemical and/or optical signal. In our case, we coupled the biosensor to the expression of anti-invertase.

pFruB - Cra (FruR) Regulation

The enterobacterial catabolite repressor/activator (Cra) protein or the FruR protein is a pleiotropic regulator that controls expression of a large number of metabolic genes in response to the flux of glycolytic intermediates in various natural biological systems. The regulator is able to interact with specifically fructose-1-phosphate and regulate the action of pFruB promoter (which is a common promoter usually found in the fructose operon) (Pereira, L.F.M. et al., 2016) (Chavarría, M et al., 2014).

Mechanism

The FruR expression is under an IPTG-induced pLac promoter (BBa_K3156000) which is induced before packaging the bacteria in our polymer inoculant. FruR is a transcription factor with an affinity for fructose-1-phosphate which is produced from the D-fructose formed after invertase action. The FruR gene (BBa_K2448009) encoding for the FruR protein prevents the transcription of the regulated promoters. pFruB (BBa_K2448017) is the promoter region following FruR and is repressed by the FruR transcription factor, in the absence of D-Fructose. This prevents any further transcription, and no anti-invertase protein is produced. If D-Fructose is present in the cell, the FruR transcription factor will bind preferentially to it and thus be inactivated. This means that the repression of the related promoter pFruB will be released, enabling the transcription of the anti-invertase protein which can then act on invertase and prevent the inversion of sucrose.

Mechanism1.png

Mathematical Model

Parameters

Parameters1.png

Differential Equations

ODE1.png

Here,

[P] is the pFruB concentration; [R] is the FruR concentration; [M] is the mCherry concentration; [A] is the anti-invertase concentration; [C1] is the concentration of the [F1P-FruR] complex; [C2] is the concentration of the [pFruB-FruR] complex (in the binding domain); [Rb] is the concentration of the domain-bound pFruB and FruR complex (Bisswanger, H., 2008); and [CB]=CNV is the approximate concentration of binding domains per cell where CN is the plasmid copy number and V is the cell volume.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2652
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2490
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 170

References

  • Park, M., Tsai, S.-L., & Chen, W. (2013), Microbial Biosensors: Engineered Microorganisms as the Sensing Machinery. Sensors 13(5), 5777-5795.
  • Pereira, L. F. M., Ferreira, V. M., Oliveira, N. G., Sarmento, P. L. V. S., Endres, L. & Teodoro, I. (2017). Sugars levels of four sugarcane genotypes in different stem portions during the maturation phase. Annals of the Brazilian Academy of Sciences. 89(2), 1231-1242.
  • Chavarr a, M., Durante-Rodr guez, G., Krell, T., Santiago, C., Brezovsky, J., Damborsky, J., & de Lorenzo, V. (2014). Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator ofPseudomonas putida. FEBS Open Bio 4(1), 377-386.