From 2012.igem.org

(Difference between revisions)

Revision as of 14:32, 21 September 2012

Module 3: Degradation

Modelling

Our cell model for this module shows the amount of laccase our bacteria produce and its action upon one type of microplastic present in the gyre, polyethylene. With this model we aimed to have an idea of how much plastic we could expect our system to degrade, which could then inform further predictive modelling around the amount of bacteria needed for Plastic Republic.

Our cell model was made in SimBiology. It consists of three compartments:

-Outside1 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

-Outside2 shows the role of laccase once outside of the cell.

Species present in the cell model

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)
Lin	0.0	Intracellular laccase
Lex	0.0	Extracellular laccase
LinmRNA	0.0	Laccase 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. The constant diffusion of POPs into the cell is essential for the continuing degradation.

At 100 seconds, the rate of degradation has risen to almost 1 PE molecule per second and we can assume that this will continue as long as the supply of POPs remains constant.

These results lead us to conclude that the initial amount of POP present is far more crucial for PE degradation than the kinetics of the laccase (Km/KCat)

Impact on experimental work

Before modelling, we were preparing to use Rhodococcus ruber strain C208 laccase, as this strain has been proven in a recent paper¹³ to degrade polyethylene. Concluding from our results that it is not enzyme activity so much as initial availability of POPs we decided to temporarily work with the most easily obtained type of laccase (from E. coli33110) and consider a later modification of our system to use C208 laccase.

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

Misc

4. Teuten E, Saquing J, Knappe D et al. (2009) Transport and release of chemicals from plastics to the environment and to wildlife. Philosophical transactions of the Royal Society of London 364: 2027-2045

11.Van A, Rochman C, Flores E, Hill K, Vargas E, Vargas S, Hoh E (2012) Persistent organic pollutants in plastic marine debris found on beaches in San Diego, California. Chemosphere 86: 258-263

12. Santo M, Weitsman R, Sivan A (2012) The role of the copper-binding enzyme - laccase - in the biodegradation of polyethylene by the actinomycete Rhodococcus ruber. International Biodeterioration & Biodegradation 208: 1-7

POPS & PLASTIC QUANTITITES

Rios L, Moore C, Jones P (2007) Persistent organic pollutants carried by synthetic polymers in the ocean environment. Marine Pollution Bulletin 54: 1230–1237

Takada H, et al (2010) Global distribution of organic micropollutants in marine plastics. Report to Algalita Marine Research Institute

Heskett M, Takada H, Yamashita R, Yuyama M, Ito M, Geok YB, Ogata Y, Kwan C, Heckhausen A, Taylor H, Powell T, Morishige C, Young D, Patterson H, Robertson B, Bailey E, Mermoz J (2012) Measurement of persistent organic pollutants (POPs) in plastic resin pellets from remote islands: Toward establishment of background concentrations for International Pellet Watch. Marine Pollution Bulletin 64: 445-448

6 Kunamneni A, Plou FJ, Ballesteros A, and Alcalde M. Laccases and their applications: A patent review. Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco, 28049 Madrid, Spain.

7. BRENDA: EC 1.10.3.2 – laccase

8. E.COLI MEMBRANE PERMEABILITY

Heskett M, Takada H, Yamashita R, Yuyama M, Ito M, Geok YB, Ogata Y, Kwan C, Heckhausen A, Taylor H, Powell T, Morishige C, Young D, Patterson H, Robertson B, Bailey E, Mermoz J (2012) Measurement of persistent organic pollutants (POPs) in plastic resin pellets from remote islands: Toward establishment of background concentrations for International Pellet Watch. Marine Pollution Bulletin 64: 445-448 is this right? This is what’s on excel

7. DIFFERENT REGULATOR SYSTEMS Wang B, Papamichail D, Mueller S, Skiena S (2007) Two Proteins for the Price of One: The Design of Maximally Compressed Coding Sequences. DOI: 10.1.1.174.6416

@@ Line 81: / Line 81: @@
 [[File:deg1.png]] In the first second of laccase production, we see polyethylene degradation beginning from around 0.6 sec (Outside2.PEdegp)
-[[File:deg2.png]] After 10 seconds, the first few molecules have been degraded.
+[[File:deg2.png]] After 10 seconds, the first few molecules have been degraded.  The constant diffusion of POPs into the cell is essential for the continuing degradation.
-[[File:deg3.png]] At 100 seconds, the rate of degradation has risen to almost 1 PE molecule per second.
+[[File:deg3.png]] At 100 seconds, the rate of degradation has risen to almost 1 PE molecule per second and we can assume that this will continue as long as the supply of POPs remains constant.
-From these results, we can conclude that
+These results lead us to conclude that the initial amount of POP present is far more crucial for PE degradation than the kinetics of the laccase (Km/KCat)
+== Impact on experimental work ==
+Before modelling, we were preparing to use <i>Rhodococcus ruber</i> strain C208 laccase, as this strain has been proven in a recent paper<sup>13</sup> to degrade polyethylene.  Concluding from our results that it is not enzyme activity so much as initial availability of POPs we decided to temporarily work with the most easily obtained type of  laccase (from <i>E. coli</i>33110) and consider a later modification of our system to use C208 laccase.
 == References ==

Team:University College London/Module 3/Modelling