|
|
(7 intermediate revisions by the same user not shown) |
Line 40: |
Line 40: |
| <div class="text"> | | <div class="text"> |
| <p style = "text-align: center;">Team UCL 2020</p> | | <p style = "text-align: center;">Team UCL 2020</p> |
− | <b>Team members</b><br> | + | <b>Student Team members</b><br> |
− | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano, Anna Su, Li Xu and Oliver Hernandez Fernandez | + | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano Garrido, Anna Su, Li Xu and Oliver Hernandez Fernandez |
| <br><br> | | <br><br> |
| | | |
Line 56: |
Line 56: |
| <div class="row"> | | <div class="row"> |
| <div class="section third" id="problem"> | | <div class="section third" id="problem"> |
− | <img src="https://static.igem.org/mediawiki/2020/4/4d/T--UCL--Poster_problem_section.png"> | + | <img src="https://static.igem.org/mediawiki/2020/b/b6/T--UCL--Poster_problem_section_final.png"> |
| | | |
| <div class="info"> | | <div class="info"> |
Line 72: |
Line 72: |
| | | |
| <div class="section third" id="motivation"> | | <div class="section third" id="motivation"> |
− | <img src="https://static.igem.org/mediawiki/2020/c/cc/T--UCL--Poster_motivation_section.png"> | + | <img src="https://static.igem.org/mediawiki/2020/1/17/T--UCL--Poster_motivation_section_final.png"> |
| | | |
| <div class="info"> | | <div class="info"> |
Line 88: |
Line 88: |
| | | |
| <div class="section third" id="idea"> | | <div class="section third" id="idea"> |
− | <img src="https://static.igem.org/mediawiki/2020/d/d3/T--UCL--Poster_idea_section.png"> | + | <img src="https://static.igem.org/mediawiki/2020/9/92/T--UCL--Poster_idea_section_final.png"> |
| | | |
| <div class="info"> | | <div class="info"> |
Line 120: |
Line 120: |
| <br> | | <br> |
| <img src="https://static.igem.org/mediawiki/2020/2/2c/T--UCL--Poster_PET_co-culture.png"> | | <img src="https://static.igem.org/mediawiki/2020/2/2c/T--UCL--Poster_PET_co-culture.png"> |
− | <p>Figure 2. Overview of the mechanism of the co-culture of <i>E. coli</i>, <i>P. putida</i> and <i>S. oneidensis</i> designed to investigate the research questions above. <i>E. coli</i> feeds on PET by secreting the PETase-MHETase fusion to the extracellular environment generating Ethylene glycol (EG) and TPA. Ethylene glycol is used to feed <i>E. coli</i>, while TPA feeds P. putida in order to maximise secretion of Lactate. The lactate generated can then feed S. oneidensis, which grows as a biofilm on the anode surface and generates a current output.</p> | + | <p>Figure 2. Overview of the mechanism of the co-culture of <i>E. coli</i>, <i>P. putida</i> and <i>S. oneidensis</i> designed to investigate the research questions above. <i>E. coli</i> feeds on PET by generating Ethylene glycol (EG) and TPA. Ethylene glycol is used to feed <i>E. coli</i>, while TPA feeds <i>P. putida</i> in order to maximise lactate secretion. The lactate generated can then feed <i>S. oneidensis</i>, which grows as a biofilm on the anode surface and generates a current output.</p> |
| </div> | | </div> |
| | | |
Line 135: |
Line 135: |
| We identified strains to express the PET degradation pathway to produce sufficient lactate to sustain the growth and electricity generation by <i>S. Oneidensis</i>. <i>E. coli</i> was engineered to degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) while <i>P. Putida</i> fully metabolizes TPA to produce lactate for <i>S. Oneidensis</i>. | | We identified strains to express the PET degradation pathway to produce sufficient lactate to sustain the growth and electricity generation by <i>S. Oneidensis</i>. <i>E. coli</i> was engineered to degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) while <i>P. Putida</i> fully metabolizes TPA to produce lactate for <i>S. Oneidensis</i>. |
| <br><br> | | <br><br> |
− | <img src="https://static.igem.org/mediawiki/2020/c/c1/T--UCL--Poster_PET_degradation_pathway.png"> | + | <img src="https://static.igem.org/mediawiki/2020/c/c1/T--UCL--Poster_PET_degradation_pathway.png" style = "width: 70%;"> |
− | <p>Figure 3. Pathway schematic representation of a model of enzymatic plastic degradation coupling microbial electricity generation. The engineered E. coli secretes PET degrading enzymes, PETase and MHETase, to degrade polyethylene terephthalate (PET) into mono-terephthalic acid (MHET) and ethylene glycol (EG) [2][3]. EG enters E. coli via transporters to be further metabolized in the native secondary pathway expressed an operon in E. coli. Metabolites in this pathway are: glycolaldehyde (GA), glycolate (GC), glycoxalate (GLA), tartronate semialdehyde (TS), and glycerate (GL). While GL enters glycolysis and gets converted into pyruvate (Pyr) to support E. coli’s biomass growth, TPA enters P. putida via TPA transporter encoded by genes from Comamonas sp. strain E6 [4]. Our engineered P. putida expresses transgenic genes to degrade TPA into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD), next into 3,4-dihydroxybenzoate (PCA) [5]. PCA enters P. putida’ s endogenous β-ketoadipate pathway. Metabolites in this pathway are: β-carboxy-cis,cis-muconate (β-CM), γ-carboxymuconolactone (γ-CML), β-ketoadipate enol-lactone (β-KG EL), β -ketoadipate (β-KG), β -ketoadipyl-CoA (β-KGCoA), succinyl-CoA (SucCoA), acetyl-CoA (AcCoA) [6]. AcCoA enters tricarboxylic acid cycle (TCA) of P. putida, generating lactate for exoelectrogen S. oneidensis. Native pathway in S. oneidensis metabolizes lactate to support biomass growth and electron production [7][8].</p> | + | <p>Figure 3. Pathway schematic representation of a model of enzymatic plastic degradation coupled with microbial electricity generation. The engineered <i>E. coli</i> secretes PET degrading enzymes, PETase and MHETase, to degrade polyethylene terephthalate (PET) into mono-terephthalic acid (MHET) and ethylene glycol (EG) (4)(5). EG enters <i>E. coli</i> via transporters to be further metabolized in the native secondary pathway expressed an operon in <i>E. coli</i> forming glycerate (GL). While GL enters glycolysis and gets converted into pyruvate (Pyr) to support <i>E. coli's</i> biomass growth, TPA enters <i>P. putida</i> via TPA transporter encoded by genes from Comamonas sp. strain E6 (6). Our engineered <i>P. putida</i> expresses transgenic genes to degrade TPA into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD), next into 3,4-dihydroxybenzoate (PCA) (7). PCA enters <i>P. putida's</i> endogenous β-ketoadipate pathway forming succinyl-CoA (SucCoA) and acetyl-CoA (AcCoA) (8). AcCoA enters the tricarboxylic acid cycle (TCA) of <i>P. putida</i>, generating lactate for exoelectrogen <i>S. oneidensis</i>. Native pathway in <i>S. oneidensis</i> metabolizes lactate to support biomass growth and electron production (9)(10). |
| + | </p> |
| | | |
| <br> | | <br> |
Line 146: |
Line 147: |
| <li>Express the TPA transporter and degradation pathway in <i>P. putida</i></li> | | <li>Express the TPA transporter and degradation pathway in <i>P. putida</i></li> |
| </ol> | | </ol> |
− | PETase and MHETase were expressed in E. coli as a fusion protein to increase the efficiency of PET degradation. We made a structural model and consulted experts prior to designing the constructs decide on the gene order of the enzymes in the fusion protein. The model showed that the MHETase-PETase construct had less steric hinderance which we hypothesized to perform better. The McGeehan group from the University of Portsmouth also verified our hypothesis as they found that the PETase-MHETase fusion could not be functionally expressed in <i>E. coli</i> due to protein aggregation (4). Therefore, we designed constructs with the individual enzymes and the two configurations of the fusion protein to test if the gene order affected extracellular secretion, functional expression or enzyme activity. | + | PETase and MHETase were expressed in E. coli as a fusion protein to increase the efficiency of PET degradation. We made a structural model and consulted experts prior to designing the constructs to decide on the gene order of the enzymes in the fusion protein. The model showed that the MHETase-PETase construct had less steric hinderance which we hypothesized to perform better. The McGeehan group from the University of Portsmouth also verified our hypothesis as they found that the PETase-MHETase fusion could not be functionally expressed in <i>E. coli</i> due to protein aggregation (11). Therefore, we designed constructs with the individual enzymes and the two configurations of the fusion protein to test if the gene order affected extracellular secretion, functional expression or enzyme activity. |
| <br><br> | | <br><br> |
| <img src="https://static.igem.org/mediawiki/2020/3/39/T--UCL--Poster_constructs.png"> | | <img src="https://static.igem.org/mediawiki/2020/3/39/T--UCL--Poster_constructs.png"> |
Line 207: |
Line 208: |
| <p>Figure 8. 1. Agent type over one a week, with time in a hours, showing active cells in blue, quiescent cells in orange, and dead cells in green. Each agent behaves as 125 cells. 2. Total current density generated by biofilm over one week</p> | | <p>Figure 8. 1. Agent type over one a week, with time in a hours, showing active cells in blue, quiescent cells in orange, and dead cells in green. Each agent behaves as 125 cells. 2. Total current density generated by biofilm over one week</p> |
| | | |
− | We observed a peak in the current density after about 48 hours at 1.2 A/m2. A steady state current density of approximately 0.0397 A/m2 was achieved lasting for over 9 days generating an average desalination rate of 0.623 L/m2/h. | + | We observed a peak in the current density after about 48 hours at 1.2 A/m2. A steady state current density of approximately 0.0397 A/m2 was achieved generating an average desalination rate of <b>0.623 L/m2/h</b> which is within the range of rates observed by Ramírez-Moreno et al. (12). |
| + | |
| + | For a lab-scale MDC with a 250 ml anode chamber and an anode surface area of 25cm2, 1.56 ml of water can be desalinated per hour. The system will have to be scaled up appropriately to desalinate enough water for irrigation. |
| + | |
| </div> | | </div> |
| </div> | | </div> |
Line 277: |
Line 281: |
| <br><br> | | <br><br> |
| <img src="https://static.igem.org/mediawiki/2020/1/1f/T--UCL--Poster_flowsheet_new.png"> | | <img src="https://static.igem.org/mediawiki/2020/1/1f/T--UCL--Poster_flowsheet_new.png"> |
− | <p>Figure 10. Flowsheet showing proposed implementation strategy of a desalination process</p> | + | <p>Figure 10. Flowsheet showing proposed implementation strategy of the desalination process</p><br> |
| + | As the FBA results showed that the co-culture and desalination have to be performed separately, the process has been split into 2 steps with an aerobic chamber for the co-culture and an anaerobic MDC chamber for electricity generation. After the co-culture, centrifugation and filtration are used to remove the cells from the outlet stream containing lactate which is then transferred to the anaerobic MDC.<br><br> |
| + | |
| + | From the CA, we found that the MDC will have to be scaled up to increase the surface area of the anode in order to desalinate enough water for irrigation. Through our research and communications with experts, we've proposed scaling up by stacking multiple MDCs or using a tubular MDC configuration. Though, this will have to be investigated further by next year's UCL iGEM team. |
| + | |
| </div> | | </div> |
| </div> | | </div> |
Line 402: |
Line 410: |
| </div> | | </div> |
| | | |
− | <br><br><p><b>Team members</b></p> | + | <br><br><p><b>Student Team members</b></p> |
− | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano, Anna Su, Li Xu and Oliver Hernandez Fernandez | + | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano Garrido, Anna Su, Li Xu and Oliver Hernandez Fernandez |
| <br><br> | | <br><br> |
| <b>Supervisors and Instructors</b><br> | | <b>Supervisors and Instructors</b><br> |
− | Dr Stefanie Frank, Dr Kenth Gustafsson, Dr Chris Barnes, Dr Darren Nesbeth, Dr Rana Khalife, Alexander Van de Steen (Postgraduate Research student), Rory Gordon (Masters Research Student) and Neythen Treolar (Postgraduate Research Student). | + | Dr Stefanie Frank, Dr Kenth Gustafsson, Dr Chris Barnes, Dr Darren Nesbeth, Dr Rana Khalife, Alexander Van de Steen (Postgraduate Research student), Rory Gordon (Masters Research Student) and Neythen Treloar (Postgraduate Research Student). |
| <br><br> | | <br><br> |
| Thanks to everyone that shared their expertise and supported us throughout the project. | | Thanks to everyone that shared their expertise and supported us throughout the project. |
Line 415: |
Line 423: |
| <li>United Nations. Water scarcity [Internet]. International Decade for Action “Water for Life” 2005-2015. 2014 [cited 2020 Oct 25]. Available from: https://www.un.org/waterforlifedecade/scarcity.shtml</li> | | <li>United Nations. Water scarcity [Internet]. International Decade for Action “Water for Life” 2005-2015. 2014 [cited 2020 Oct 25]. Available from: https://www.un.org/waterforlifedecade/scarcity.shtml</li> |
| <li>Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS One. 2014 Dec 10;9(12).</li> | | <li>Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS One. 2014 Dec 10;9(12).</li> |
| + | <li>Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science (80- ) [Internet]. 2016 Mar 11;351(6278):1196 LP – 1199. Available from: http://science.sciencemag.org/content/351/6278/1196.abstract</li> |
| + | <li>Salvador M, Abdulmutalib U, Gonzalez J, Kim J, Smith AA, Faulon J-L, et al. Microbial Genes for a Circular and Sustainable Bio-PET Economy. Genes (Basel) [Internet]. 2019 May 16;10(5):373. Available from: https://pubmed.ncbi.nlm.nih.gov/31100963</li> |
| + | <li>Hosaka M, Kamimura N, Toribami S, Mori K, Kasai D, Fukuda M, et al. Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6. Appl Environ Microbiol [Internet]. 2013/08/02. 2013 Oct;79(19):6148–55. Available from: https://pubmed.ncbi.nlm.nih.gov/23913423</li> |
| + | <li>Johnson CW, Beckham GT. Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin. Metab Eng [Internet]. 2015;28:240–7. Available from: http://www.sciencedirect.com/science/article/pii/S1096717615000075</li> |
| + | <li>Wang J-Y, Zhou L, Chen B, Sun S, Zhang W, Li M, et al. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity. Sci Rep [Internet]. 2015 Dec;5(1):18456. Available from: http://www.nature.com/articles/srep18456</li> |
| + | <li>Kane AL, Brutinel ED, Joo H, Maysonet R, VanDrisse CM, Kotloski NJ, et al. Formate Metabolism in Shewanella oneidensis Generates Proton Motive Force and Prevents Growth without an Electron Acceptor. Silhavy TJ, editor. J Bacteriol [Internet]. 2016 Apr;198(8):1337–46. Available from: https://jb.asm.org/content/198/8/1337</li> |
| + | <li>Luo S, Guo W, H. Nealson K, Feng X, He Z. 13C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells. Sci Rep [Internet]. 2016 Aug;6(1):20941. Available from: http://www.nature.com/articles/srep20941</li> |
| <li>Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci [Internet]. 2020 Oct 13 [cited 2020 Nov 9];117(41):25476–85. Available from: www.pnas.org/cgi/doi/10.1073/pnas.2006753117</li> | | <li>Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci [Internet]. 2020 Oct 13 [cited 2020 Nov 9];117(41):25476–85. Available from: www.pnas.org/cgi/doi/10.1073/pnas.2006753117</li> |
| + | <li>Ramírez-Moreno M, Rodenas P, Aliaguilla M, Bosch-Jimenez P, Borràs E, Zamora P, et al. Comparative Performance of Microbial Desalination Cells Using Air Diffusion and Liquid Cathode Reactions: Study of the Salt Removal and Desalination Efficiency. Front Energy Res [Internet]. 2019 Dec 5 [cited 2020 Oct 24];7:135. Available from: https://www.frontiersin.org/article/10.3389/fenrg.2019.00135/full</li> |
| </ol> | | </ol> |
| </div> | | </div> |