Team:University College London/Module 1/Modelling

From 2012.igem.org

(Difference between revisions)
(→Modelling)
(→References)
 
(32 intermediate revisions not shown)
Line 6: Line 6:
== Modelling ==
== 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.  
+
As our detection module is so closely tied up with our degradation and aggregation modules, 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 degradation system.  
[[File:detectionnet.jpg]]
[[File:detectionnet.jpg]]
-
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.
+
Our cell model for detection was created in SimBiology.  This MathWorks software is a MATLAB extension that allows specification of the species, reactions, and compartments that make up a system - in our case, the cell and the external environment - and to run simulations on the specified system.  For this module, we are interested in detection response, which we represent here as GFP production. The model is composed of two 'compartments', which in SimBiology represent separate reaction systems:
-
-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
+
1. ''''External'''': 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 (10<sup>5</sup> to 10<sup>6</sup> the specificity to our system.)
 +
 
 +
 
 +
2. ''''Cell'''': NahR is a constitutively produced mRNA product (R7). 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 GFP (R5, R6).  GFP is expressed in the cell or degrades (R9).
==Species ==
==Species ==
 +
{| class="bigtable"
{| class="bigtable"
|-
|-
-
! Species!! Initial value (molecules) !! Notes  
+
! Species!! Initial value (molecules) !! Notes & Assumptions
|-
|-
| PE|| 0.044 || Polyethylene found in North Pacific Gyre (value per cubic metre)<sup>1,2</sup>
| PE|| 0.044 || Polyethylene found in North Pacific Gyre (value per cubic metre)<sup>1,2</sup>
Line 26: Line 29:
| POPex || 0.0 || Persistent organic pollutants (ex = extracellular) that are not adhered to plastic surface
| 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 surface<sup>3</sup>
+
| PEPOPex || 9.24E-5 || Persistent organic pollutants (ex = extracellular) that are adhered to the plastic surface<sup>6</sup>
|-
|-
| POPin || 0.5 || Persistent organic pollutants (in = intracellular) assumed from <i>E. coli</i> membrane permeability <sup>4</sup>
| POPin || 0.5 || Persistent organic pollutants (in = intracellular) assumed from <i>E. coli</i> membrane permeability <sup>4</sup>
Line 36: Line 39:
| POPinNahRpSal || 0.0 || Complex of the above molecule and pSal (promoter that induces laccase transcription)
| POPinNahRpSal || 0.0 || Complex of the above molecule and pSal (promoter that induces laccase transcription)
|-
|-
-
| GFP || 0.0 || Intracellular laccase
+
| GFP || 0.0 || Green fluorescent protein
|-
|-
| GFPmRNA || 0.0 || GFP mRNA product
| 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
 
|}
|}
Line 49: Line 48:
{| class="bigtable"
{| class="bigtable"
|-
|-
-
! Number !! Reaction !! Reaction rate (molecules/sec) !! Notes  
+
! Number !! Reaction !! Reaction rate (molecules/sec) !! Notes & Assumptions
|-
|-
-
| R1 || PE + POPex ↔ PEPOPex || Forward: 1000 <br /> Backward: 1 || Pops have 1000 to 10000 times greater tendency to adhere to plastic than float free in the ocean<sup>5</sup>
+
| R1 || PE + POPex ↔ PEPOPex || Forward: 10000 <br /> Backward: 1 || Pops have 10000 to 100000 times greater tendency to adhere to plastic than float free in the ocean<sup>5</sup>
|-
|-
-
| R2 || POPex ↔ POPin || Forward: 0.6 <br /> Backward: 0.4 || Based on membrane permeability<sup>4</sup>: diffusion gradient
+
| R2 || POPex ↔ POPin || Forward: 0.6 <br /> Backward: 0.4 || Rate is based on membrane permeability<sup>4</sup> and diffusion gradient
|-
|-
-
| R3 || POPin + mRNA.Nahr → POPin.mRNA.Nahr || Forward: 1 <br /> Backward: 0.0001 || Based on the assumption that the chemical structure/size of POPs is similar to salycilate<sup>6</sup>.  Salycilate binds to the NahR mRNA product, which complex then binds to the pSal promoter.
+
| R3 || POPin + mRNA.Nahr ↔ POPin.mRNA.Nahr || Forward: 1 <br /> Backward: 0.0001 || Based on the assumption that the chemical structure/size of POPs is similar to salycilate<sup>6</sup>.  Salycilate binds to the NahR mRNA product, which complex then binds to the pSal promoter.
|-
|-
-
| R9 || 0 → mRNA.Nahr || Forward: 0.088 <br /> Backward: 0.6 || Transcription rate of NahR in molecules/sec (for NahR size 909 bp<sup>7</sup>, transcription rate in E.coli 80bp/sec<sup>8</sup>) under constitutive promoter control
+
| R4 || POPinmRNANahr ↔ POPinmRNANahr.Psal || Forward: 78200 <br /> Backward: 0.191 <sup>9</sup> || NahR to pSal binding based on the assumption that POP-NahR binding has no effect on NahR-pSal binding
|-
|-
-
| R4 || POPinmRNANahr → POPinmRNANahr.Psal || Forward: 78200 <br /> Backward: 0.191 <sup>9</sup> || NahR to pSal binding based on the assumption that POP-NahR binding has no effect on NahR-pSal binding
+
| R5 || POPexmRNANahr.Psal → GFP.mRNA|| 0.11 || Transcription rate of GFP in molecules/sec (for GFP size 720bp<sup>10</sup>, transcription rate in E.coli 80bp/sec<sup>8</sup>)
|-
|-
-
| R5 || POPexmRNANahr.Psal → Lin.mRNA|| 0.054 || Transcription rate of Laccase in molecules/sec (for laccase size 1500 bp<sup>10</sup>, transcription rate in E.coli 80bp/sec<sup>8</sup>)
+
| R6 || GFP.mRNA → GFP|| 0.084 || Translation rate of GFP in molecules/sec (for GFP size 240aa<sup>10</sup>, translation rate in E.coli 20aa/sec<sup>8</sup>)
|-
|-
-
| R6 || Lin.mRNA → Lin|| 0.04 || Translation rate of Laccase in molecules/sec (for laccase size 500 aa<sup>10</sup>, translation rate in E.coli 20aa/sec<sup>8</sup>)
+
| R7 || 0 → mRNA.Nahr || Forward: 0.088 <br /> Backward: 0.6 || Transcription rate of NahR in molecules/sec (for NahR size 909 bp<sup>7</sup>, transcription rate in E.coli 80bp/sec<sup>8</sup>) under constitutive promoter control
|-
|-
-
| R11 || Lin → Lindegp || 0.03 || Degradation rate of laccase<sup>11</sup> must be taken into account due to suboptimal conditions
+
| R9 || GFP.mRNA → 0 || 0.03 || Degradation rate of GFP mRNA product<sup>11</sup> must be taken into account due to suboptimal conditions
-
|-
+
-
| R7 || Lin → Lex || Forward: 0.9 <br /> 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 periplasm<sup>12</sup>, which is able to degrade polyethene.
+
-
|-
+
-
| R8 || Lex → PEdegp || Vm: 0.01 <br /> 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 polyethylene<sup>13</sup> 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 literature<sup>14</sup>
+
|}
|}
== Results ==
== Results ==
-
We ran three simulations in SimBiology, each over a different timespan:
+
[[File:gfpgraph.png]]
-
 
+
-
[[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:deg3.png]] At 100 seconds, the rate of degradation has risen to almost 1 PE molecule per second.
+
-
From these results, we can conclude that
+
Results from model simulations show that GFP is expressed upon detection of persistent organic pollutants, such as polyethylene, in line with expectations. The rate of increase in fluorescence levels eventually decreases, with a maximal asymptotic expression being reached. With this in mind and satisfied that degradation is working as expected ''in silico'', we extended the model to create the [[Team:University_College_London/Module_3/Modelling|cell model for degradation]] and show how polyethylene is degraded by Laccase proteins and how sensitive it is to changes within biochemical rates.
== References ==
== References ==
Line 90: Line 79:
2. Andrady AL (2011) Microplastics in the marine environment. <i>Marine Pollution Bulletin</i> 62: 1596-1605
2. Andrady AL (2011) Microplastics in the marine environment. <i>Marine Pollution Bulletin</i> 62: 1596-1605
-
3. To follow
+
4. Kay J, Koivusalo M, Ma X, Wohland T, Grinstein S (2012) Phosphatidylserine Dynamics in Cellular Membranes. <i>Molecular Biology of the Cell</i>
-
 
+
-
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. <i>Environ. Sci. Technol.</i> 35: 318-324
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. <i>Environ. Sci. Technol.</i> 35: 318-324
Line 104: Line 91:
9. Park H, Lim W, Shin H (2005) In vitro binding of purified NahR regulatory protein with promoter Psal. <i>Biochimica et Biophysica Acta</i> 1775: 247-255
9. Park H, Lim W, Shin H (2005) In vitro binding of purified NahR regulatory protein with promoter Psal. <i>Biochimica et Biophysica Acta</i> 1775: 247-255
-
10. Laccase size: http://partsregistry.org/Part:BBa_K729002
+
10. http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522
-
 
+
-
11. To follow
+
-
 
+
-
12. Young K, Silver LL (1991) Leakage of periplasmic enzymes from envA1 strains of Escherichia coli. <i>J Bacteriol.</i> 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.  <i>African Journal of Microbiology Research</i> 6: 1147-1157.  DOI: 10.5897/AJMR11.1257
+
-
 
+
-
 
+
-
 
+
-
[[File:detectiongraph.jpg]]
+
-
1. GFP size: http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522
+
11. Kushner S (2002) mRNA Decay in <i>Escherichia coli</i> Comes of Age. <i>J Bacteriol.</i> 184: 4658-4665
-
2. NahR size: http://www.xbase.ac.uk/genome/azoarcus-sp-bh72/NC_008702/azo2419;nahR1/viewer
 
{{:Team:University_College_London/templates/foot}}
{{:Team:University_College_London/templates/foot}}

Latest revision as of 13:33, 26 September 2012

Contents

Module 1: Detection

Description | Design | Construction | Characterisation | Modelling | Conclusions

Modelling

As our detection module is so closely tied up with our degradation and aggregation modules, 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 degradation system.

Detectionnet.jpg

Our cell model for detection was created in SimBiology. This MathWorks software is a MATLAB extension that allows specification of the species, reactions, and compartments that make up a system - in our case, the cell and the external environment - and to run simulations on the specified system. For this module, we are interested in detection response, which we represent here as GFP production. The model is composed of two 'compartments', which in SimBiology represent separate reaction systems:


1. 'External': 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 (105 to 106 the specificity to our system.)


2. 'Cell': NahR is a constitutively produced mRNA product (R7). 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 GFP (R5, R6). GFP is expressed in the cell or degrades (R9).

Species

Species Initial value (molecules) Notes & Assumptions
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 surface6
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 Green fluorescent protein
GFPmRNA 0.0 GFP mRNA product

Reactions taking place in the model

Number Reaction Reaction rate (molecules/sec) Notes & Assumptions
R1 PE + POPex ↔ PEPOPex Forward: 10000
Backward: 1 		Pops have 10000 to 100000 times greater tendency to adhere to plastic than float free in the ocean5
R2 POPex ↔ POPin Forward: 0.6
Backward: 0.4 		Rate is based on membrane permeability4 and 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.
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 → GFP.mRNA 0.11 Transcription rate of GFP in molecules/sec (for GFP size 720bp10, transcription rate in E.coli 80bp/sec8)
R6 GFP.mRNA → GFP 0.084 Translation rate of GFP in molecules/sec (for GFP size 240aa10, translation rate in E.coli 20aa/sec8)
R7 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
R9 GFP.mRNA → 0 0.03 Degradation rate of GFP mRNA product11 must be taken into account due to suboptimal conditions

Results

Gfpgraph.png

Results from model simulations show that GFP is expressed upon detection of persistent organic pollutants, such as polyethylene, in line with expectations. The rate of increase in fluorescence levels eventually decreases, with a maximal asymptotic expression being reached. With this in mind and satisfied that degradation is working as expected in silico, we extended the model to create the cell model for degradation and show how polyethylene is degraded by Laccase proteins and how sensitive it is to changes within biochemical rates.

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

4. Kay J, Koivusalo M, Ma X, Wohland T, Grinstein S (2012) Phosphatidylserine Dynamics in Cellular Membranes. Molecular Biology of the Cell

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. http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522

11. Kushner S (2002) mRNA Decay in Escherichia coli Comes of Age. J Bacteriol. 184: 4658-4665


Retrieved from "http://2012.igem.org/Team:University_College_London/Module_1/Modelling"