Part:BBa_K3512012

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.

Mathematical Model

Parameters

Differential Equations

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); and [Pb] is the concentration of pFruB bound to the binding domain (Bisswanger, H., 2008)(#citation4); [Rb] is the concentration of the domain-bound pFruB and FruR complex (Bisswanger, H., 2008)

Molecular Modelling

We docked FruR and Fructose-1-Phosphate using AutoDock.

We also docked the modelled FruR-pFruB complex with Fructose-1 phosphate using AutoDock. We observed that there were no significant interactions taking place which meant that there is a conformational change induced by pFruB on FruR. We can see that the major fructose binding pocket undergoes a conformational change (shown by the dis-alignment of the initial blue beta sheet and the new red beta sheet) leading to a changed beta sheet structure.

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.