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Team:University College London/Module 1/Modelling
From 2012.igem.org
Contents |
Module 1: Detection
Description | Design | Construction | Characterisation | Modelling | Conclusions
Modelling
As our detection module is so closely tied up with our degradation module, our cell model for this module examines the hypothetical expression of GFP on detection of POPs. This allows us to assess the efficacy of detection as a stand-alone module before we look at its performance when incorporated into our system.
Note that this model is very similar to our cell model for degradation. This model, however, looks only at detection response - modelled here by GFP production - and ignores the production and action of laccase.
-External represents the constant association and disassociation of persistent organic pollutants (POPs) that takes place in the ocean.
-Cell represents the reactions taking place inside the cell
Species
| Species | Initial value (molecules) | Notes |
|---|---|---|
| PE | 0.044 | Polyethylene found in North Pacific Gyre (value per cubic metre)1,2 |
| POPex | 0.0 | Persistent organic pollutants (ex = extracellular) that are not adhered to plastic surface |
| PEPOPex | 9.24E-5 | Persistent organic pollutants (ex = extracellular) that are adhered to the plastic surface3 |
| POPin | 0.5 | Persistent organic pollutants (in = intracellular) assumed from E. coli membrane permeability 4 |
| mRNANahR | 0.0 | NahR mRNA product |
| POPinNahR | 0.0 | Complex of the above two molecules |
| POPinNahRpSal | 0.0 | Complex of the above molecule and pSal (promoter that induces laccase transcription) |
| GFP | 0.0 | Intracellular laccase |
| GFPmRNA | 0.0 | GFP mRNA product |
| Ldegp | 0.0 | Laccase that degrades due to suboptimal conditions and malformed laccase that cannot carry out polyethylene degradation |
| PEdegp | 0.0 | Polyethylene degraded by laccase |
Reactions taking place in the model
| Number | Reaction | Reaction rate (molecules/sec) | Notes |
|---|---|---|---|
| R1 | PE + POPex ↔ PEPOPex | Forward: 1000 Backward: 1 | Pops have 1000 to 10000 times greater tendency to adhere to plastic than float free in the ocean5 |
| R2 | POPex ↔ POPin | Forward: 0.6 Backward: 0.4 | Based on membrane permeability4: diffusion gradient |
| R3 | POPin + mRNA.Nahr → POPin.mRNA.Nahr | Forward: 1 Backward: 0.0001 | Based on the assumption that the chemical structure/size of POPs is similar to salycilate6. Salycilate binds to the NahR mRNA product, which complex then binds to the pSal promoter. |
| R9 | 0 → mRNA.Nahr | Forward: 0.088 Backward: 0.6 | Transcription rate of NahR in molecules/sec (for NahR size 909 bp7, transcription rate in E.coli 80bp/sec8) under constitutive promoter control |
| R4 | POPinmRNANahr → POPinmRNANahr.Psal | Forward: 78200 Backward: 0.191 9 | NahR to pSal binding based on the assumption that POP-NahR binding has no effect on NahR-pSal binding |
| R5 | POPexmRNANahr.Psal → Lin.mRNA | 0.054 | Transcription rate of Laccase in molecules/sec (for laccase size 1500 bp10, transcription rate in E.coli 80bp/sec8) |
| R6 | Lin.mRNA → Lin | 0.04 | Translation rate of Laccase in molecules/sec (for laccase size 500 aa10, translation rate in E.coli 20aa/sec8) |
| R11 | Lin → Lindegp | 0.03 | Degradation rate of laccase11 must be taken into account due to suboptimal conditions |
| R7 | Lin → Lex | Forward: 0.9 Backward: 0.1 | Our laccase is a periplasmic enzyme, therefore most of it is released in the periplasm. However, we assumed leakage of 20 percent based on suboptimal conditions in the periplasm12, which is able to degrade polyethene. |
| R8 | Lex → PEdegp | Vm: 0.01 Km: 0.114 | Michaelis-Menten kinetics is used to represent degradation of polyethylene. As the literature values for the Km and Kcat for the polyethylene by laccase are not yet obtained experimentally for the original starin that was observed being able to degrade polyethylene13 we made an assumption that degradation of polyethylene is similar to that of lignin (due to polymeric nature of both), values for both Km and Kcat were taken from the literature14 |
Results
We ran three simulations in SimBiology, each over a different timespan:
In the first second of laccase production, we see polyethylene degradation beginning from around 0.6 sec (Outside2.PEdegp)
After 10 seconds, the first few molecules have been degraded.
At 100 seconds, the rate of degradation has risen to almost 1 PE molecule per second.
From these results, we can conclude that
References
1. Goldstein M, Rosenberg M, Cheng L (2012) Increased oceanic microplastic debris enhances oviposition in an endemic pelagic insect, Biology Letters 10.1098
2. Andrady AL (2011) Microplastics in the marine environment. Marine Pollution Bulletin 62: 1596-1605
3. To follow
4. To follow
5. Mato Y, Isobe T, Takada H, Kanehiro H, Ohtake C, Kaminuma T (2001) Plastic Resin Pellets as a Transport Medium for Toxic Chemicals in the Marine Environment. Environ. Sci. Technol. 35: 318-324
6. https://2011.igem.org/Team:Peking_S/project/wire/harvest
7. http://www.xbase.ac.uk/genome/azoarcus-sp-bh72/NC_008702/azo2419;nahR1/viewer
8. http://kirschner.med.harvard.edu/files/bionumbers/fundamentalBioNumbersHandout.pdf
9. Park H, Lim W, Shin H (2005) In vitro binding of purified NahR regulatory protein with promoter Psal. Biochimica et Biophysica Acta 1775: 247-255
10. Laccase size: http://partsregistry.org/Part:BBa_K729002
11. To follow
12. Young K, Silver LL (1991) Leakage of periplasmic enzymes from envA1 strains of Escherichia coli. J Bacteriol. 173: 3609–3614
13. To follow (c208 ref??)
14. 5. Ding Z, Peng L, Chen Y, Zhang L, Gu Z, Shi G, Zhang K (2012) Production and characterization of thermostable laccase from the mushroom, Ganoderma lucidum, using submerged fermentation. African Journal of Microbiology Research 6: 1147-1157. DOI: 10.5897/AJMR11.1257
1. GFP size: http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522
2. NahR size: http://www.xbase.ac.uk/genome/azoarcus-sp-bh72/NC_008702/azo2419;nahR1/viewer
