"
Team:ITB Indonesia/Modeling
From 2014.igem.org
Degradation Module
The degradation reaction of PET
PET+LC Cutinase→Ethylene Glycole +Terephtalic Acid+Cutinase
- Cutinase
The amount of LC-Cutinase enzyme is due to the enzyme production rate by Escherichia coli and the degradation of LC-Cutinase. The enzyme (LC-Cutinase) production occurs constitutively (did not need any activator and produced continuously). At the t-time when LC-Cutinase reach the maximum quantity, the Escherichia coli productivity will be decreased because of Inclusion body. Inclusion body is protein which is produced by miss-folded protein. The amount of inclusion body will be increases. When the inclusion body reach the K quantity, it will be repress the LC-Cutinase production because the inclusion body become a inducer for PibPab promoter that could drive Tet Represor gene expression. Our degradation module contain Tet Operator that could be repressed by Tet Represor. Our PibPab promoter also could drive the Amil Cp gene expression that release blue chromoprotein. Based on that phenomenon, the production of LC-Cutinase was modeled using Hill Function with the repressor factor.
- Inclusion Body
When the Escherichia coli start to produce many of cutinase enzyme at maximum concentration, it start become stress and begin to producing a miss-folded protein called inclusion body. As long as the producing of enzyme cutinase in this condition, the producing of inclusion body will be much. At this time the inclusion body act as the repressor that stop the production of cutinase and also will be the activator of reporter gen (Amil CP). This activity shown by the picture above. The model expression of inclusion body was modeled using Hill Function.
The assumption of the model:
We do not include the factor of cell division of bacteria
Cutinase
Inclusion Body inducer
Reporter gen (Amil CP) expression
Ethylene Glycole amount (as a product of PET degradation)
PET degradation
Figure 1 shown the graph of cutinase production, inclussion body production and blue protein expression. Figure 2 shown the the close up graph and Figure 3 shown the PET degradation and ethylene glicol product profil by the time of gene expression. Inclussion bodies are reflactile aggreates of protease-resistant misfolded protein that often occur in recombinant bacteria upon gratutious overexpression of cloned genes. In biotechnology, the formation of Ibs represents a main obstacle for protein production since even favouring high protein yields. Aggregation of recombinant proteins is probably due to limiting amount of chaperones when recombinant gene expression is directed at high leves. When the ammount of protein production is maximal, we assume that inclusion body will be release. Inclussion body could drive the expression of Tet represor and AmilCp as shown in figure 1. When the inclussion body could be degraded by the cell, the PibPab promoter will be off and there are no TetR and amilCP expression. In that condition we assume that the the cutinase expression will be increassing until reach some value that could release the inclusion body, than the expression will be represed. In figure 2, we could observe the osilation graph. From the figure 3, we could see that by the time of PET degradation the ethylene glicol product that could be released is increase.
- References
- Carrio, M.M and A. Villaverde. 2002. Construction and Deconstruction of Bacterial Inclusion Bodies. Journal of Biotechnology 96. 3-12
- Chapman, and Halu/CRC, “AN INTRODUCTION TO SYSTEMS BIOLOGY Design Principle of Biological Circuit”
- Fink, Anthony L. 1998. Protein Aggregation: folding aggregates, inclusion bodies and amyloid. © Current Biology Ltd ISSN 1359-0278
