Module 3: Degradation

Description | Design | Construction | Characterisation | Modelling | Results | Conclusions

Modelling

Building upon our earlier cell model for detection of polyethylene, our cell model for the degradation module aimed to show the amount of laccase our bacteria produce and its role in the degradation of 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. This would then be used in further predictive modelling to calculate the amount of bacteria needed for the construction of Plastic Republic. The following diagram shows the degradation system, with each reaction R_i and each DNA species explained below.

Our cell model was made in SimBiology, which is a MATLAB extension used to run simulations of the reactions in a specified system. It shows the following reactions taking place in three 'compartments', with each DNA species and reaction explained in further detail below:

1. Outside1: in this compartment we see the association, adherence, and dissociation of persistent organic pollutants (POPs) from polyethylene (PE) (R1). We rely on the POPs to induce our degradation system, increasing from 10⁵ to 10⁶ the specificity to our system.

2. Cell: NahR is a constitutively produced mRNA product (R9). When POP diffuses into the cell (R2), it forms a complex with NahR (R3) which then binds to the pSal promoter (R4) to induce production of laccase (R5, R6). Around 20% of this laccase diffuses outside of the cell (R7), the rest degrades (R10).

3. Outside2: in this compartment, the external laccase degrades PE (R9). Some of this external laccase is degraded (R11).

Species present in the cell model

Reactions taking place in the model

Results

We ran three simulations in SimBiology, each over a different timespan to analyse how laccase production changed over time. In the first second of laccase production, we see polyethylene degradation beginning from around 0.6 sec:

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:

From these results, we can see that our bacteria will degrade polyethylene. However, what these graphs can't show are the factors affecting degradation. For this reason, we decided to run a sensitivity analysis on our model.

Sensitivity analysis

We used SimBiology's sensitivity analysis tool to compute the sensitivity of the cell to variations in each model parameter and the initial amount of each DNA species. The aim is to find out how each of these impacts on the final concentrations of degraded PE and laccase.

The results show that the amount of PE degraded is dependent on the kinetics of the laccase reaction (see the paramters labelled R8.k17 and R8.k19 in the upper bars in the diagram below, which correspond to the Vm and Km values respectively of laccase). On the other hand, the amount of laccase degraded outside the cell is most dependent on the translation rate of laccase (see the parameter R6.k20 in the lower bars of the diagram). In addition, it also moderately affected by the rate of diffusion of laccase outside of the cell (see R7.p10) and therefore on the amount of laccase which ends up outside of the cell (Lexdegp).

Impact on experimental work

We initially believed that it was the amount of POPs present in the environment, rather than the specific activity of the enzyme used, which would have the greatest effect on the amount of polyethylene degraded. Thus, although we had initially identified Rhodococcus ruber C208 laccase strain¹³ to use in the degradation of polyethylene, we now decided to use a laccase which was easier to obtain (Ganoderma lucidum).

Following the results of the sensitivity analysis, we realised that using a laccase with greater specific activity on PE would have a measurable impact on the amount of plastic our system can degrade. Hence, future modifications to our system will 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. Kushner S (2002) mRNA Decay in Escherichia coli Comes of Age. J Bacteriol. 184: 4658-4665

12. Young K, Silver LL (1991) Leakage of periplasmic enzymes from envA1 strains of Escherichia coli. J Bacteriol. 173: 3609–3614

13. 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

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

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