Team:SDU-Denmark
editors highlights
ENERGY MADE BEAUTIFUL With the PowerLeaf, iGEM SDU is introducing a novel solution for long-term storage of solar energy, thus becoming an alternative to solar cells, without using environmentally harmful resources. We aim to accomplish this through the creation of a device visually shaped to resemble a plant leaf, thereby providing a nature-in-city ambience. The team invested heavily in public engagement and collaborations to investigate how the device hypothetically could be implemented into an urban environment. From a technical perspective, the bacterial solar battery is composed of an energy storing unit and an energy converting unit. The energy storing unit is defined by a genetically engineered Escherichia coli, that fixates carbon dioxide into the chemically stable polymer cellulose, while the energy converting unit uses genetically engineered Escherichia coli to consume the stored cellulose. Electrons retrieved from this process, are transferred to an anode by optimised nanowires, thereby creating an electrical current. Last but not least, the energy storing unit has a light-dependent system, which activates dormancy during nighttime to reduce energy lost by metabolism.
A green project, a green wiki, and a great performance in the iGEM Goes Green initiative! Green just got greener. Introduction Welcome to our wiki! We are the University of Southern Denmark's iGEM team and we have been waiting in great anticipation for the chance to tell you our story.
At the Giant Jamboree 2017 we succeeded in getting a Gold Medal and were nominated 'Best Energy Project'. Bronze Medal Requirements Register and Attend – Our team applied on the 30th of March 2017 and got accepted the 4th of May 2017. We had an amazing summer and are looking forward to attending the Giant Jamboree! Silver Medal Requirements Validated Part/Contribution – We created the part BBa_K2449004, containing a cellobiose phosphorylase, Cep94A. This enzyme enables Escherichia coli to survive on cellobiose, which we validated by growth experiments. The data obtained in these experiments are presented in the Demonstration & Results section. Gold Medal Requirements Integrated Human Practices – For the development and implementation of the device, we reached out to and remained in contact with city planners from our hometown throughout our project. This regarded advice and conversations on anything from the possible design, value, safety, use, placement, and plastic type of our device. The ideas generated from these conversations, were integrated in our overall project. Last but not least, we focused on demonstrating this process on our wiki in order to inspire future iGEM teams. A Global Challenge In the world of today, it is becoming increasingly important to ensure a sustainable future Green Growth Papers (Myriam Linster). Material Resources, Productivity and the Environment. 2013.. Not just for our generation, but especially for the generations to come, as their possibilities should not be limited by our choices.
Our solution, is the development of a green and renewable technology, which offers new advantages to the field of sustainable energy. There are currently certain limitations to the existing options for renewable energy, namely the intermittency and the diluteness problem Alexandre Chagnes JS. Global Lithium Resources and Sustainability Issues. Lithium Process Chemistry: Elsevier; June 2015. p. pp.1-40.. The intermittency problem describes the discontinuous energy production, along with inefficient storage. On the other hand, the diluteness problem is characterised as the resource-demanding production of technical devices, such as solar cells and batteries. This means that a lack of resources eventually could eliminate some of the current forms of green technology. As such, we need to introduce a new and sustainable approach to green energy to ensure the continuation of our beautiful world for the coming generations.
In a Local Environment We are a team of young adults raised to be aware of climate changes and the potential limitations to our ways of life. As a generation that appreciates open source and shared information, we have been encouraged to constantly challenge the ideas of yesterday. With this in mind, we decided the best solution to the eventual energy crisis would be to seek out experts and the general public, even children, in order to rethink the current notion that the only way to save our planet, is to compromise our living standards.
Inspiration Our early ideas were reviewed after attending the Danish Science Festival, where we met several young minds with creative and inspiring ideas. The children came to our workshop with their parents to learn about bacteria, the history of GMO, ethics, and iGEM. They attended our “Draw-a-Bacteria”-competition, where they designed their own bacteria, some even with detailed stories. From this the children taught us a thing or two about the endless possibilities of GMO. See a selection of their amazing drawings here. “Well, if it can be thought, it can be done, a problem can be overcome” E.A. Bucchianeri, Brushstrokes of a Gadfly The vision for our bacterial solar battery is to combine two aspects, energy storage and energy conversion, by which we will produce a new and improved type of solar battery. We have named this vision The PowerLeaf. The PowerLeaf consist of two chambers that will be referred to as the outer chamber or energy storing unit and the inner chamber or energy converting unit. The complete system will be combined into a single device containing a compartment for each of the two units. Details about the construction and device will be discussed in the Integrated Practices section.
It will then be up to future iGEM teams to continue on the development of the PowerLeaf. We would love to see our project become a reality one day, and so we have created a special page for future iGEM teams, which includes suggestions for a further development of the project.
Project & Results Our device is composed of two units, an energy storing unit and an energy converting unit, each divided into systems, all of which have been given a symbol to help you navigate throughout the wiki. Energy Storing Unit Dormancy System Carbon Fixation Cellulose Biosynthesis Energy Converting Unit Breakdown of Cellulose Extracellular Electron Transfer In the Project Design section, you will first be given a short introduction to the background, followed by the approach of that system, before you move on to the next system. Once you reach the next section of the wiki, Demonstration & Results, you will be guided through the performed experiments and the derived conclusions. To make things easier for you, we have continued to use the above symbols throughout our wiki.
Project Overview Introduction Cyanobacteria contain signal transduction systems, thereby making them capable of sensing and responding to light Bussell AN, Kehoe DM. Control of a four-color sensing photoreceptor by a two-color sensing photoreceptor reveals complex light regulation in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(31):12834-9.. This ability gives the organisms the opportunity to adapt and optimize their metabolism to a circadian rhythm. Photoreceptors in the plasma membrane, of which phytochromes are especially abundant and well described, are responsible for this property Vierstra RD, Davis SJ. Bacteriophytochromes: new tools for understanding phytochrome signal transduction. Seminars in cell & developmental biology. 2000;11(6):511-21.. In 2004, the UT Austin iGEM team made a light response system consisting of a photoreceptor combined with an intracellular indigenous regulator system Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2.. EnvZ and OmpR make up the two-component system naturally found in E. coli. The photoreceptor known as Cph1 was isolated from the cyanobacteria Synechocytis PCC6803. Cph1 has functional combination sites, which combined with the kinase EnvZ form a two-domain receptor, known as Cph8. Activation of Cph8 is mediated by the chromophore phycocyanobilin, PCB, that is sensitive to red light with maximal absorbance at 662 nm Lamparter T, Esteban B, Hughes J. Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure. European journal of biochemistry. 2001;268(17):4720-30..
Figure 1. Left: Red light activates PCB, which in turn inactivates the photoreceptor complex Cph8, preventing gene expression from the OmpR-regulated promoter. Right: In absence of light, PCB is inactive, which enables the Cph8 to phosphorylate the transcription factor OmpR. This promotes gene expression from the OmpR-regulated promoter.
The photocontrol device can be used to regulate a toxin-antitoxin system, enabling the implementation of a light-dependent dormancy system. A toxin-antitoxin system is composed of two gene products, a cytotoxin and an antitoxin, the latter which neutralises the the toxic effect caused by the toxin.
In E. coli K-12 the cytotoxin RelE and antitoxin RelB comprise such a system Gotfredsen M, Gerdes K. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Molecular microbiology. 1998;29(4):1065-76.. Expression of the cytotoxin RelE inhibits translation in the cells, due to its ability to cleave mRNA found in the A-site of the ribosome. RelB neutralises the toxic effect of RelE through interaction between the two proteins. Whether the cell lies dormant in response to expression of RelE depends on the ratio of antitoxin RelB and RelE present in the cell. Several studies have shown that RelB and RelE form a complex with RelB:RelE stoichiometry of 2:1 Overgaard M, Borch J, Gerdes K. RelB and RelE of Escherichia coli form a tight complex that represses transcription via the ribbon-helix-helix motif in RelB. Journal of molecular biology. 2009;394(2):183-96.Overgaard M, Borch J, Jorgensen MG, Gerdes K. Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular microbiology. 2008;69(4):841-57.. When the RelB:RelE stoichiometric-ratio is lowered to 1:1, studies show that RelB is not able to protect the cells against the RelE-caused translational inhibition Overgaard M, Borch J, Jorgensen MG, Gerdes K. Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular microbiology. 2008;69(4):841-57.. For further information about the light-dependent dormancy system, read here.
Modelling
Modelling of the RelE-RelB System is Essential to Avoid Irrevocable Dormancy Controllable dormancy is a feature that holds the potential to be applied in many different situations. However, inducing dormancy and bringing the bacteria back to a metabolic active state is like balancing on a tightrope, and to establish the basis of future implementations, the properties of this system would have to be investigated further. In an endeavour to provide this basic knowledge, stochastic modelling utilising the Gillespie algorithm was performed in an attempt to prognosticate the system and simulate the interactions between the toxin and antitoxin.
The toxin RelE is inhibited by the antitoxin RelB through complex formation, and both proteins interact with their promoter in a feedback mechanism.
To consolidate the model, the capacity of the toxin-antitoxin system was assessed in an experiment, as the controllability of the dormancy system was studied through manual regulation of RelE and RelB expression. Figure 2. Left: The time required for the bacteria to enter dormancy varies with the expression level of RelB. The percentage of dormant bacteria, defined as containing RelE amounts above 40 molecules per cell as a function of time in minutes. Right: Only one of the tested configurations, RelB2:50-RelE:35, causes the bacteria to regain their activity within the modelled time. The percentage of dormant bacteria, defined as containing RelE amounts above 15 molecules per cell as a function of time in minutes. The data is based on the simulation of 1000 independent bacteria.
The simulated data revealed, that when enhanced RelE production is implemented, in order to induce dormancy in E. coli, the effect come easily. However, implementation of RelB expression is also found necessary to ensure that the bacteria are able to enter an active state again. Approach
In 2004 the Austen and UCSF iGEM team created a device sensitive to light, laying the foundation for the Coliroid project. In this project, the system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. As tight regulation is required for the RelE-RelB system Tashiro Y, Kawata K, Taniuchi A, Kakinuma K, May T, Okabe S. RelE-Mediated Dormancy Is Enhanced at High Cell Density in Escherichia coli. J Bacteriol. 2012;194(5):1169-76., modelling of the toxin-antitoxin system is essential. The impact of different RelE-RelB expression levels was simulated by modelling. Using the results obtained by this modelling, a hypothetical working system-design was devised.
For further information about our approach, read here. Figure 3. The final design of the light-dependent dormancy system. RelE under control of the OmpR-regulated promoter on the chromosome or a low copy vector, RelB under the control of the pBAD promoter on a low copy vector, and the photocontrol device controlled by the PenI-regulated promoter on a high copy vector. Approach Balancing Bacterial Dormancy Requires Accurate Regulation of the System Figure 1. All three components of the light-dependent dormancy system cloned into one high copy plasmid.
The Photocontrol Device was Placed under Control of a Constitutive Promoter Figure 2. Cultures of E. coli TOP10. From left to right: WT, the weak promoter, BBa_J23114, the two medium promoters, BBa_J23110, and BBa_J23106, and the strong promoter, BBa_J23102.
On the basis of the data obtained by fluorescence microscopy, the strong constitutive promoter BBa_J23102, was chosen to control the photocontrol device. For still inexplicable reasons the photocontrol device emerged difficult to clone with the strong constitutive promoter. Although the molecular cloning of these parts was optimised several times very few successful clonings were accomplished, and the few times correct assembly was obtained, the BioBrick was not reproducibly purifiable. By sequencing, it was deduced that a region of the plasmid containing both the promoter and the BioBrick prefix had vanished. To circumvent this inconvenient cloning two other promoters, that are constitutive in E. coli, were examined, namely the PenI-regulated, BBa_R0074, and the Mnt-regulated, BBa_R0073, promoters. By performing fluorescence microscopy on composite parts of the promoters controlling yellow fluorescent protein (YFP), BBa_I6102, and BBa_I6103, respectively, the expression levels were assessed. The obtained results revealed that the PenI-regulated promoter facilitated very strong expression of the marker gene, whereas the expression controlled by the Mnt-regulated promoter was noticeably lower. Based on these findings, the photocontrol device was placed under the control of the PenI-regulated promoter instead of the strong constitutive promoter from the constitutive promoter family. As it turned out, this cloning likewise emerged difficult. After several attempts, it was decided to focus on the other aspects of the dormancy system.
Figure 3. The principle behind the recombination. By PCR, the two flanking sequences are assembled with the fragment containing the chloramphenicol resistance gene (camR), the OmpR-regulated promoter, and the relE gene. The flanking sequences are homologous to part of the chromosome around the bacterial attachment site (attB), enabling the homologous recombination.
Using this approach, it was attempted to assemble the OmpR-regulated promoter controlling RelE with the flanking regions by PCR. The assembly of the fragment derived from the plasmid with either one of the flanking fragments was achieved. However, the assembly of the complete chromosomal insertion fragment containing all three segments emerged problematic. Several attempts with numerous process optimisations were performed, but unfortunately without success. To circumvent this, another attachment site was chosen. The assembly of the complete fragment by PCR was achieved and chromosomal insertion by electroporation was attempted several times, but in vain. In consideration of the fact that RelE is a bacterial toxin, the cloning of this gene was inconvenient. The model indicated, that overexpression of the antitoxin RelB would bypass this difficulty. As the homologous recombination of this fragment emerged such a challenging task, it was decided to focus on the conduct of the OmpR-regulated promoter on different plasmids, as this was evidently the more convenient approach of the general implementation of the OmpR-regulated promoter.
Figure 4. The final design of the light-dependent dormancy system. RelE under control of the OmpR-regulated promoter on the chromosome or a low copy vector, RelB under the control of the pBAD promoter on a low copy vector, and the photocontrol device controlled by the PenI-regulated promoter on a high copy plasmid. Project Overview Introduction
Carbon fixation in autotrophic organisms is responsible for the net fixation of 7×1016 g carbon annually Berg IA. Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Applied and Environmental Microbiology. 2011;77(6):1925-36.. Six different pathways related to carbon fixation have been discovered, but the most widespread of these, is the Calvin-Benson-Bassham (CBB) cycle found in photosynthetic eukaryotes, e.g. plants and algae, as well as in photo- and chemosynthetic bacteria B. Bowien MG, R. Klintworth, U. Windhövel. Metabolic and Molecular Regulation of the CO2-assimilating Enzyme System in Aerobic Chemoautotrophs. Microbial Growth on C1 Compounds: Proceedings of the 5th International Symposion. 1st ed. Institute for Microbiology, Georg-August-University Göttingen, Federal Republic of Germany: Martinus Nijhoff Publishers; 1987.. Out of the eleven enzymes needed for the Calvin cycle, only three are heterologous to E. coli, namely: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase (PRK). By the concurrent heterologous expression of the three genes encoding these enzymes, E. coli can be engineered to perform the full Calvin cycle. Figure 4. A simplified illustration of the Calvin cycle, with the enzymes heterologous to E. coli and their respective substrates and products shown. The carboxysome is a microcompartment utilised by many chemoautotrophic bacteria, including cyanobacteria, as a CO2 accumulating mechanism to increase carbon fixation efficiency. This organelle-like polyhedral body is able to increase the internal concentrations of inorganic carbon by 4000-fold compared to the external concentration Mangan NM, Brenner MP. Systems analysis of the CO(2) concentrating mechanism in cyanobacteria. eLife. 2014;3.. One type of carboxysome is the ɑ-carboxysome, which consists of a proteinaceous outer shell composed of six different shell proteins designated CsoS1ABCD and CsoS4AB. This shell encloses RuBisCo, the shell associated protein (CsoS2), and the enzyme carbonic anhydrase (CsoS3). In the proteobacteria Halothiobacillus neapolitanus, these genes are clustered into the cso operon. The carbonic anhydrase converts HCO3-, which diffuses passively into the carboxysome, to CO2, thereby driving the continued diffusion of HCO3- into the microcompartment Mangan NM, Brenner MP. Systems analysis of the CO(2) concentrating mechanism in cyanobacteria. eLife. 2014;3.. The increased CO2 concentration in the vicinity of RuBisCo increases the rate of carbon fixation by saturating the RuBisCo enzyme and increasing the CO2 to O2 ratio, enabling carboxylation to dominate over oxygenation Mangan NM, Brenner MP. Systems analysis of the CO(2) concentrating mechanism in cyanobacteria. eLife. 2014;3.. The shell associated protein is essential for the biogenesis of the ɑ-carboxysome Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, et al. Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life (Basel, Switzerland). 2015;5(2):1141-71.. Figure 5. An illustration of the ɑ-carboxysome. The shell proteins CsoS1ABC and CsoS4AB enclose the enzymes RuBisCo and carbonic anhydrase. For the Calvin cycle to proceed, energy in the form of ATP and electrons carried by NADPH are required. The photosystems are complexes in photosynthesising organisms that can supply this by photophosphorylation. To engineer E. coli to do photosynthesis, 13 genes is needed for the assembly of chlorophyll a and 17 genes for the assembly of photosystem II, which needs to be heterogeneously expressed. An alternative process in which a diverse array of phototrophic bacteria and archaea harvest energy from light, is through a retinal-containing protein called proteorhodopsin that catalyses the light-activated proton efflux across the cell membrane, and thereby drive ATP synthesis. Opposed to the photosystems, the proteorhodopsin is anoxygenic and generates no NADPH, which is crucial for the Calvin cycle to proceed Walter JM, Greenfield D, Bustamante C, Liphardt J. Light-powering Escherichia coli with proteorhodopsin. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(7):2408-12.. For further information about the carbon fixation, read here.
Approach In order to engineer E. coli in the outer chamber to turn atmospheric CO2 into cellulose, the carbon first needs to be fixated by the bacteria. This requires the heterologous expression of the genes encoding the three enzymes RuBisCo, SBPase, and PRK. Furthermore, the implementation of the carboxysome from the cso operon can increase the levels of carbon fixation. The 2014 Bielefeld iGEM team had worked with a similar approach in their project. In an endeavour to optimise the carbon fixation process, our project build upon their experiences. The assembly of the individual parts into a composite part, BBa_K2449030, was achieved, however, the cloning of these parts with a promoter proved problematic. Consequently, it was decided to prioritise other aspects of the project and therefore keep this part theoretical henceforth. For further information about our approach, read here.
Project Overview Introduction
Bacterial cellulose is one of the most abundant biopolymers produced by different species of gram-negative bacteria, especially by Acetobactors. Glucoacetobacter xylinus is a bacterial species that produces cellulose of high quality in large quantitiesLin, SP., Loira Calvar, I., Catchmark, J.M. et al. Cellulose (2013) 20: 2191.. Cellulose is produced from the resource glucose-6-phosphate. This phosphorylated glucose is a key intermediate in the core carbon metabolism of bacteria given its importance in glycolysis, gluconeogenesis and the pentose phosphate pathway Joanne Willey LS, Christopher J. Woolverton. Prescott’s Microbiology. 9th edition 2014.. Even though the pathway, where glucose and glucose-6-phosphate is converted into cellulose, only includes few steps, it requires a great amount of energy. Not only does the cell spend energy on forming UDP-glucose for cellulose biosynthesis, it also uses glucose, which otherwise would have contributed to generation of ATP Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, et al. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(24):E3431-40..
Figure 6. The AcsAB dimer synthesises cellulose nanofibers. AcsC and AcsD mediate the secretion and formation of an interconnected cellulose pellicle. Other genera, including some E. coli strains, secrete cellulose as a component of their biofilm. Even though cellulose biosynthesis is intrinsic to E. coli, the quantity of the production is incomparable to cellulose biosynthesis in G. xylinus. Indigenously, E. coli is not capable of degrading cellulose into a metabolisable energy source Gao D, Luan Y, Wang Q, Liang Q, Qi Q. Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase. Microbial Cell Factories. 2015;14:159.. However, if this structural and water-holding polymer is enzymatically degraded, first into cellobiose and then to glucose residues, the cellulose polymer is a potent source of energy Arai T, Matsuoka S, Cho HY,
Yukawa H, Inui M, Wong SL, et al. Synthesis of Clostridium cellulovorans
minicellulosomes by intercellular complementation. Proceedings of the National
Academy of Sciences of the United States of America. 2007;104(5):1456-60..
Approach
To link the two bacterial compartments of the PowerLeaf, an efficient way to store the harvested energy was required. Through research, we found that storing the chemical energy in cellulose would be a suitable approach, since this is a polysaccharide that bacteria normally are unable to degrade Gao D, Luan Y, Wang Q, Liang Q, Qi Q. Construction of cellulose-utilizing Escherichia coli based on a secretable cellulase. Microbial Cell Factories. 2015;14:159.. After looking into earlier iGEM projects it was found that the
2014 project Aqualose from Imperial College London, had worked with optimisation of cellulose biosynthesis in E. coli. Our aim was to enhance cellulose biosynthesis in E. coli MG1655, which naturally secretes small amounts of cellulose as a part of its biofilm Gualdi L, Tagliabue L, Bertagnoli S, Ierano T, De Castro C, Landini P. Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli. Microbiology (Reading, England). 2008;154(Pt 7):2017-24.. This would be achieved by the cloning of plasmids containing the cellulose synthase operon acsABCD, utilising the two parts BBa_K1321334 and BBa_K1321335, constructed by Imperial College London 2014. This would enhance the cellulose biosynthesis and thereby optimise the energy outcome of the entire system in our project.
Due to cloning difficulties, it was decided to prioritise other aspects of the project and therefore keep this part theoretical henceforth. For further information about the cellulose biosynthesis approach, read here.
Project Overview Introduction
Cellulose is a natural biopolymer used for a vast variety of biological purposes and it is most commonly found in plants, where it serves as the main structural component. Since plants are primary producers, many organisms of the Earth’s ecosystems have adapted accordingly Lynd
LR, Weimer PJ, van Zyl WH, Pretorius IS. Microbial Cellulose Utilization:
Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews.
2002;66(3):506-77.. One of the key evolutionary features for the primary consumers was the development of the ability to degrade cellulose into glucose, which could then be used as a cellular fuel. A simple organism, able to efficiently do so, is the Cellulomonas fimi, which converts cellulose to glucose in a two-step process, with cellobiose as the intermediate Jung
SK, Parisutham V, Jeong SH, Lee SK. Heterologous Expression of Plant Cell Wall
Degrading Enzymes for Effective Production of Cellulosic Biofuels. Journal of Biomedicine and Biotechnology. 2012;2012..
Breakdown of Cellulose to Cellobiose Figure 7. Degradation of the β-1,4-linkages in cellulose mediated by the enzymes endo-β-1,4-glucanase and exo-β-1,4-glucanase, thereby creating cellobiose.
The α-Hemolysin Transport System Figure 8. The enzymes encoded by the cenA and cex genes are linked to HlyA. HlyB recognises HlyA and initiates transportation of the HlyA-tagged protein from the cytosol to the extracellular fluid.
Uptake of Cellobiose
Degradation of Cellobiose to Glucose Figure 9. Phosphorylation of the β-1,4-linkages in cellobiose by the enzyme cellobiose phosphorylase, thereby producing D-glucose and α-D-glucose-1-phosphate.
Approach
Cellulose to Cellobiose Figure 10. BioBrick, containing the genes cenA, cex, hlyB, and hlyD controlled by PenI-regulated promoters.
Cellobiose to Glucose Figure 11. BioBrick comprising cep94A controlled by a LacI-regulated promoter. This part was cloned into both a high and low copy vector.
Project Overview Introduction
Microbial Fuel Cell Figure 12. A microbial fuel cell utilising glucose as substrate. The glucose is consumed to protons, electrons, and CO2. The electrons are transferred to the anode while the protons diffuse over the proton exchange membrane. A gradient causes the electrons to flow through an external load to the cathode, which generates an electrical current.
Under aerobic conditions, the generated NADH will deliver its electron as part of the electron transfer chain, thereby returning to its oxidised form NAD+. Under anaerobic conditions the electron transport chain will be unable to continue, which will cause the generated NADH to accumulate, and as a consequence, the concentration of available NAD+ for glycolysis will decrease. This will drive the cell to carry out other metabolic pathways, such as fermentation, in order to maintain its ATP levels. Instead, the accumulating NADH generated under anaerobic conditions, can be utilised to drive an electrical current by depositing the retrieved electrons to an anode coupled with an appropriate cathode. The cathode catalyst in an MFC will usually catalyse the reaction of 2 H+ + ½ O2 per H2O. The transfer of electrons from NADH to the anode can be executed in three different ways, as shown in Figure 13: redox shuttles, direct contact electron transfer, and bacterial nanowires Logan BE, Hamelers B, Rozendal R, Schroder U, Keller J, Freguia S, et al. Microbial fuel cells: methodology and technology. Environmental science & technology. 2006;40(17):5181-92.Khanal YLaSK. Microbial Fuel Cells, Capter 19. In: Khanal. EbYLaSK, editor. Bioenergy: Principles and Applications. First Edition ed: Published 2017 by John Wiley & Sons, Inc.; 2016.. Figure 13. Three different ways to transfer electrons from microorganisms to an anode. a) Transfer of electrons to the anode using a redox shuttle (Med). Two different types of redox shuttles exit: One going through the membrane and another receiving electrons from membrane proteins. b) Transfer of electrons to the anode by direct contact. c) Electrons are carried from the inside of the cell, directly to the anode through nanowires.
The redox shuttles use extracellular electron mediators, which hold the advantage of not being limited by the surface area of the anode, although it is restricted by the slow diffusion of the extracellular mediators. The direct contact electron transfer is, in contrast to the redox shuttles, strongly limited by the surface area of the anode, but the membrane bound cytochromes that are in direct contact with the anode, rapidly deliver the electrons. Bacterial nanowires are known to efficiently transfer electrons, as for the direct contact electron transfer. However, bacterial nanowires are not as strictly limited by the surface area of the anode as the direct contact electron transfer. This is due to the ability of bacterial nanowires to form complex networks of interacting nanowires in biofilm, thereby efficiently transferring electrons from distant microbes to the anodeKhanal YLaSK. Microbial Fuel Cells, Capter 19. In: Khanal. EbYLaSK, editor. Bioenergy: Principles and Applications. First Edition ed: Published 2017 by John Wiley & Sons, Inc.; 2016..
Bacterial Nanowires Figure 14. The electrons from NADH are transferred to menaquinone (MQ), reducing it to menaquinol (MQH2), the inner membrane-associated MaCA cytochrome receives the electrons and reduces the periplasmic triheme cytochromes (PpcA-PpcE). The electrons are mediated to the outer membrane-associated cytochromes, OmcB and OmcE, and further transferred to cytochromes on the pili Morgado L, Fernandes AP, Dantas JM, Silva MA, Salgueiro CA. On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochemical Society transactions. 2012;40(6):1295-301..
The electrical conductivity of the nanowires in G. sulfurreducens can be optimised by exchanging endogenous pilA with heterologous pilA rich in aromatic amino acids. Tan Yang et. al 2017 Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, et al. Expressing the Geobacter metallireducens pilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity. mBio. 2017;8(1).heterogeneously expressed pilA from G. metallireducens in G. sulfurreducens, which increased the electrical conductivity of the recombinant bacteria 5000-fold. This optimisation holds great potential in the development of highly efficient bacterial strains for MFCs. With the intention of optimising an MFC, G. sulfurreducens is a lot easier to work with than G. metallireducens Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, et al. Expressing the Geobacter metallireducens pilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity. mBio. 2017;8(1)., since G. metallireducens has a longer generation time.
Approach
Originally, it was intended to implement bacterial nanowires from G. sulfurreducens into E. coli. Through research, it was found that the Bielefeld iGEM team from 2013 had come to the conclusion, that this task was too comprehensive to undertake in the limited time of an iGEM project. However, a different approach was deviced, as postdoc Oona Snoeyenbos-West suggested us to use G. sulfurreducens as the model organism for our MFC.
Figure 15. The linear DNA fragment intended for homologous recombination into G. sulfurreducens.
Project Overview Determination of Noise Levels in Constitutive Promoter Family Members
Fluorescence microscopy and flow cytometry revealed that a strong constitutive promoter was suitable for the expression of the photocontrol device. For further information about the experiments and a proposed cause for the increasing noise level, read here. Expression Level of the Mnt- and Penl-Regulated Promoters
The PenI-regulated promoter was chosen to control the expression of the photocontrol device, as it mediated a stronger expression than the Mnt-regulated promoter. Figure 16. Fluorescence microscopy of YFP under the control of PenI-regulated and Mnt-regulated promoters on pSB1C3 in E. coli MG1655 at OD600=0.3-0.5. From the data obtained, it was evident that the PenI-regulated promoter mediated a substantially higher level of YFP expression than the Mnt-regulated promoter, as seen in Figure 16. Furthermore, the PenI-regulated promoter displayed a notably lower level of noise. On the basis of these results, the PenI-regulated promoter was chosen to control the expression of the photocontrol device.
From the data obtained, it was evident that the PenI-regulated promoter mediated a substantially higher level of YFP expression than the Mnt-regulated promoter, as seen in Figure 16. Furthermore, the PenI-regulated promoter displayed a notably lower level of noise. On the basis of these results, the PenI-regulated promoter was chosen to control the expression of the photocontrol device.
Leaky Expression by the OmpR-Regulated Promoter on Different Vectors
Leaky expression by the OmpR-regulated promoter is reduced when cloned into a low copy vector compared to a high copy vector. Figure 17. Fluorescence microscopy of RFP controlled by the OmpR-regulated promoter on a high copy vector in E. coli strain SØ928 ΔompR. On the basis of this finding, the relE gene controlled by the OmpR-regulated promoter required a low copy plasmid or insertion into the chromosome. Protein expression of RFP in pSB1C3 with a copy number of 100-300 plasmids per cell, and pSB3K3 with a copy number of 10-12 plasmids per cell, was studied by flow cytometry. As for the determination of noise levels in the weak, BBa_J23114, and strong BBa_J23102 constitutive promoters, the experiment was carried out in both LB medium and M9 minimal medium, the latter supplemented with 0.2% glycerol. In the LB medium, selection was carried out by the addition of 30 µg/mL chloramphenicol, 30 µg/mL kanamycin, or 50 µg/mL ampicillin, depending on the resistance, and for M9 minimal medium, the concentrations used were 60 µg/mL chloramphenicol, 60 µg/mL kanamycin, and 100 µg/mL ampicillin. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. Expression levels in both E. coli MG1655 and E. coli MG1655 ΔompR were studied to determine the baseline of the leaky expression not influenced by intrinsic pathways including the OmpR transcription factor. Figure 18. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the the OmpR-regulated promoter on the high copy vector, pSB1C3, and the low copy vector, pSB3K3, in MG1655 WT and ΔompR MG1655 strain. All fluorescence levels were measured relative to the negative control WT E. coli MG1655, and the weak and strong constitutive promoters are included as references. Standard error of mean is shown, but are in several cases indistinguishable from the graph. Fluorescence levels in the two different media display similar behavior, as seen in Figure 18. The main difference observed, was that the decrease in fluorescence over time was faster in LB medium than in M9 minimal medium, in concordance with the observations made in previous experiments. On a general level, the data revealed, that MG1655 cloned with the POmpR-RFP reporter system on the high copy vector exhibited a fluorescence level, equivalent to that mediated by the strong constitutive promoter. On the low copy vector, the POmpR-RFP reporter system yielded a fluorescence level comparable to the gene expression mediated by the weak constitutive promoter. On the other hand, expression levels in the MG1655 ΔompR strain were markedly reduced compared to MG1655, indicating that pathways including the transcription factor OmpR interfere with RFP expression under these conditions. Again, the fluorescence levels observed for the POmpR-RFP reporter system on the low copy vector were distinctly lower than for the high copy vector. Transposon Hotspot Formation in LacI-Regulated lambda pL Hybrid Promoter Reporter System
During cloning, formation of a transposon hotspot between the LacI-regulated lambda pL hybrid promoter and a reporter system was observed, making this promoter inapt for the dormancy system Induction and Subsequent Inhibition of the pBAD Promoter
Expression by the pBAD promoter can be regulated tightly by induction and subsequent inhibition. Figure 19. YFP fluorescence levels in E. coli MG1655 transformed with the pBAD-YFP reporter system on pSB3K3. Left: Cultures with the inducer arabinose added. Right: Cultures not induced with arabinose. Both cultures were split up at OD600=0.1, designated time 0, and the inhibitor glucose was added to one half of each culture. Images were obtained at 0, 30, 60, and 120 minutes. This experiment made it clear, that gene expression controlled by the pBAD promoter is both inducible and repressible as required when cloned into the low copy vector pSB3K3. Consequently, the pBAD promoter was found to be suitable for controlling gene expression of the antitoxin RelB in the implemented dormancy system. For these experiments, the promoters controlling an RFP reporter system were cloned into E. coli strain MG1655 on pSB1A2. The cultures were grown in LB medium containing 50 µg/mL ampicillin and examined by fluorescence microscopy. For this purpose an Olympus IX83 with a photometrics prime camera was used, set to an excitation at 550 nm, emission at 573-613 nm, and exposure time at 200 ms, and cultures were taken at OD600=0.3-0.5. For flow cytometry, both LB medium with 50 µg/mL ampicillin and M9 medium with 100 µg/mL ampicillin were used. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. Figure 1. Fluorescence microscopy of RFP under the regulation of four members of the Anderson promoter collection, (BBa_J23114, BBa_J23110, BBa_J23106 and BBa_J23102) on pSB1A2 in E. coli MG1655 at OD600=0.3-0.5. BBa_J23114 displayed a low and noisy expression of RFP, whereas both BBa_J23106 and BBa_J23110 showed a more uniform expression of medium intensity. The strongest promoter, BBa_J23102, exhibited a high level of consistent RFP expression The noise levels observed by fluorescence microscopy led to the design of an experiment, by which the cell population could be quantitatively studied. The change in expression levels and noise during growth of cell populations were tested for the weak and strong constitutive promoters, for which flow cytometry was used to assess the expression of the RFP reporter system. First, the cell populations were studied in LB media with 50 µg/mL ampicillin, thereby maintaining similar conditions as for the fluorescence microscopy experiment. All cultures were prepared from overnight cultures, obtaining a starting OD600=0.005.
Figure 2. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the weak and strong constitutive promoters were measured relative to the negative control WT E. coli MG1655. Samples where measured in technical replicates and standard error of mean is shown, but are in several cases indistinguishable from the graph.
The data obtained in this experiment revealed that the strong promoter displayed a higher basal level of RFP expression than the weak promoter. Moreover, both promoters exhibited similar decrease in fluorescence over time, as seen in Figure 2, indicating that a considerable portion of the cell populations for both promoters lost their ability to fluoresce during growth, with one population containing around 60% non-fluorescent bacteria, clearly divided from the fluorescent portion. As expected, the strong promoter displayed a high level of RFP expression, roughly 500-fold of the non-fluorescent MG1655 control at 1 hour. A 10-fold decrease in fluorescence was observed from 1 hour to 4 hours. The RFP expression mediated by the weak promoter exhibited a similar decrease in fluorescence over time, though with measured fluorescence levels 5 times lower than for the strong promoter. Figure 3. The average plasmid loss measured as non-resistant CFU as a percentage counts for the ampicillin resistance of the weak and strong promoter, as well as for chloramphenicol and kanamycin resistance controls. Samples were obtained for spread-plating at 12 hours after incubation start.
From this test, it is evident that antibiotic resistance is reduced for the ampicillin-resistance carrying bacteria, compared to chloramphenicol and kanamycin resistant cultures.
This consolidates the hypothesis, that the bacteria transformed with the weak and strong promoter controlling RFP expression lose their plasmids throughout growth. However, it was further observed, that the plasmid loss in bacteria carrying the strong constitutive promoter was markedly higher than for bacteria carrying the weak constitutive promoter. This could be due to the fact that RFP in high concentrations is toxic for the cells, resulting in an increased pressure to lose the plasmid.
The photocontrol device genes were, on the basis of the above mentioned promoter experiments, placed under control of the strong promoter BBa_J23102. However, the observed plasmid loss should be taken into account, and compensated for, e.g. by continuous supply of antibiotic or substitution of the ampicillin resistance cassette.
Furthermore, as a result of the irregular expression levels controlled by the constitutive promoters, it was decided to place the relB gene under control of a tightly regulated uniform promoter, as the modelling had established the necessity of this.
During the cloning of GFP, BBa_I13504 under control of the LacI-regulated lambda pL hybrid promoter, BBa_R0011, it was observed that the part yielded three bands when digested with EcoRI and PstI, see Figure 1. One band was observed at 2000 bp, corresponding to the pSB1C3 backbone, one band at around 950 bp, correlating with the expected length for the part, and a third band with a length around 1200 bp. Figure 1. Agarose gel showing the digestion of the composite part by EcoRI and PstI. Three bands where observed. The bands at 2000 bp and 950 bp corresponded to the backbone and inserted part respectively. The third band at 1200 bp was unexpected.
To ascertain the reason for the band with the unaccountable length, the part was sequenced using the iGEM primers VF2 and VR. The sequencing revealed a repeated sequence inserted between the LacI-regulated, lambda pL hybrid promoter and the GFP reporter, encoding a transposable element. The cloning was repeated, with a similar result. Given that a similar part , differing only in the RBS sequence, had been successfully assembled and tested before by the Kyoto iGEM team from 2010. It was hypothesised, that this particular assembly could cause the formation of a transposon hotspot. Project Overview Several experiments were conducted to showcase our bacterium’s ability to grow on cellulose as the only carbon source, including Congo red screening assays and growth experiments. In addition, we performed Coomassie stained SDS-PAGE to visualise the expression of the cellulose degrading enzymes cep94A, cex and cenA. Detection of Cellulase Secreted by Type I Secretion System was Inconclusive
To assess our cellulose degrading strains of E. coli, we performed a screening with carboxymethyl cellulose (CMC) Congo red agar plates. Congo red binds to cellulose molecules, thus making it possible to visualise the breakdown of the Congo red bound cellulose. Consequently, if the cellulose molecules are degraded the red color will fade. As seen in Figure 20 the experiment did not show any visible difference between the bacteria containing cellulase secreting BioBricks and the controls, suggesting that the type I secretion system is not working as intended. Figure 20. E. coli cultures containing seven different BioBricks plated on a Congo red plate containing the water soluble cellulose derivative CMC. Cellobiose phosphorylase on pSB1C3 with LacI promoter (cep94A), exoglucanase on pSB1C3 with PenI promoter (cex), endoglucanase on pSB1C3 with PenI promoter (cenA), endoglucanase + exoglucanase on pSB1C3 with PenI
promoter (cenA+cex), endoglucanase + secretion system on pSB1C3 with
PenI promoter (cenA+SS), endoglucanase + exoglucanase + secretion system on pSB1C3
with PenI promoter (cenA and cex + SS) and LacI promoter + RBS on pSB1C3 (negative control). Measurable Expression of Cellobiose Phosphorylase Figure 21-Right shows that E. coli containing cep94A produces a protein with a molecular weight of approximately 90kDa, which is not produced by a negative control that only contains a PenI regulated promoter. This is in accordance with the expected weight of the protein expressed by the BioBrick containing the cep94A at 92.7 kDA. The protein can be found in the cell lysate regardless of whether the plasmid has a medium or high copy backbone, which is shown in Figure 21-Left. Figure 21. Left: Coomassie stained SDS-PAGE showing cell lysate and media from E. coli containing the cellobiose phosphorylase (cep94A), with Lacl promoter on the medium copy plasmid pSB3K3 and the high copy plasmid pSB1C3.The LacI promoter was induced with IPTG. Right: Coomassie stained SDS-PAGE of whole cell lysates of E.coli transformed with cep94A with a Lacl promoter compared to a negative control containing LacI + RBS. Approximate reference molecular weights are indicated to the left. Secretion of the cellulases, endoglucanase and exoglucanase, was examined on a SDS-PAGE, with samples taken from the media isolated from an overnight culture. From the SDS-PAGE at Figure 22 it was not possible to identify any proteins at the expected mass of endoglucanase and exoglucanase (47 kDa and 51 kDA, respectively), that were differentially expressed compared to control strains. However, a positive control should have been included for more conclusive results. Figure 22. Coomassie stained SDS-PAGE wíth Media
isolated from five E.coli strains, from left to right: Endoglucanase
and exoglucanase + secretion system with PenI promoter (cenA & cex + SS),
endoglucanase + secretion system with PenI promoter (cenA + SS), endoglucanase + exoglucanase with PenI promoter (cenA and cex), endoglucanase with PenI promoter (cenA), exoglucanase with PenI promoter cex. All plasmids were on pSB1C3 plasmid.
Approximate molecular weights are indicated to the left.
Cellobiose Phosphorylase Makes E. coli Able to Grow on Cellobiose The first growth experiments that were conducted included aeration of the bacteria at 200 rpm. In these experiments shown in Figure 23, we do not observe any major differences in growth between the strains containing the cep94A gene, the bglX gene and the control. Figure 23. Growth experiment with the aeration at 200 rpm. On the x-axis time in hours and OD600 on a logarithmic y-axis with standard deviation. All the experiments were conducted at once in the same water bath at 37°C and the data shown is an average of technical triplicates. Measurements were performed on E. coli MG1655 with inserted cep94A encoding cellobiose phosphorylase on pSB1C3 backbone, bglX encoding
beta-glucosidase and a negative control with LacI promoter+RBS. All the strains were growing on cellobiose+casamino acids referred as Cellobiose and just casamino acids referred as none. The Lacl promoters were induced with IPTG. The cultures were left at 37°C overnight without aeration. Increased growth was measured on the spectrophotometer at OD600 in the flasks with E. coli expressing the cep94A gene, indicating that cep94A coding cellobiose phosphorylase gives E. coli the ability to live on cellobiose. To be certain that it was the cep94A that ensured E. coli was living on cellobiose and not a contamination, we conducted a test with only a control (LacI and RBS) and cep94A, with measurements at 48 and 72 hours. This experiment revealed that cultures containing the cep94A are able to grow without aerating to an OD600 around 0.9, as shown in Figure 24. The control living only on casamino acid only grew to an OD600 around 0.2 and did not change between the two measurements. Figure 24. Column diagram with standard deviation visualising the difference in OD600 between a negative
control which is E. coli transformed with Lac-promoter + RBS
on pSB1C3(green) and E.coli
transformed with cep94A which encodes the cellobiose phosphorylase(yellow) after respectively 48 and 72 hours without aeration. The Lacl promoters was induced with IPTG. As we intend to break down cellulose to energy, we need to combine the BioBricks, meaning a strain containing cep94A on the pSB3K3 backbone and the secretion system with the endo- and exoglucanase on pSB1C3. This led us to repeating the first growth experiment with a slow aeration (40 rpm), just to make sure the media was mixed. The results are shown in Figure 25, which shows that E. coli is able to grow on cellobiose when transformed with the cep94A/cellobiose phosphorylase on the high copy backbone pSB1C3 and unable to grow when transformed with bglX or with cep94A on the medium copy backbone pSB3K3. Due to no growth on cep94A on the medium copy backbone pSB3K3, in the strain living on cellulose did not grow either. After 72 hours it was visually displayed that only cep94A transformed E. coli grew, which is illustrated in Figure 26. Figure 25. The four figures all have time in hours on the x-axis and OD600 on a logarithmic y-axis with standard deviation. All the experiments were conducted at once in the same water bath at 37°C and the data shown is an average of
technical triplicates, taken every eight hours. Measurements was performed on
E. coli MG1655 with inserted cep94A encoding cellobiose phosphorylase on pSB1C3 backbone and PenI promoter, cep94A encoding cellobiose phophorylase on pSB3K3 backbone and PenI promoter, bglX encoding beta-glucosidase with a Lacl promoter and a negative control with LacI promoter+RBS. Measurements was done on all strains growing on cellobiose+casamino acids referred as Cellobiose and just casamino acids referred as none. An addition parallel growth experiment was done on E. coli MG1655 with transformation of secretion system and cellulases on pSB1C3
backbone and cep94A encoding cellobiose phosphorylase on a pSB3K3 backbone, these measurements were done on strains growing on cellulose+casamino acids (Cellulose) and just casamino acids (none). The Lacl promoters was induced with IPTG. (A) The eight curves shown in this figure signifies all the different BioBricks supposed to degrade cellobiose, as well as a negative control. (B) The type I secretion system with cellulases ability to grow with cellulose as sole carbon
source. (C) cep94A on the pSB1C3 plasmid compared to the pSB3K3 plasmid. (D) cep94A compared to bglX. Figure 26. Cuvettes containing 2 mL of six different samples from the growth experiment from Figure 25. The cuvettes contain from left to right, cep94A in cellobiose media, cep94A with no carbon source, a negative control in cellobiose media, a negative control with no carbon source, bglX in cellobiose media and bglX with no carbon source. All plasmids was on the pSB1C3 backbone. In conclusion, we created a BioBrick containing cep94A coding for the cellobiose phosphorylase, which made it possible for the E. coli to live on cellobiose. It was not possible to show or detect whether the bacteria could live on cellulose as intended, and we detected no expression of the endoglucanase and exoglucanase. Project Overview As shown in the design of our device, the nanowires transfer electrons from the bacteria to the anode. Optimisation of the nanowires was examined using homologous recombination to exchange the endogeneous G. sulfurreducens PilA gene with the higher conducting G. metallireducens pilA gene, rich in aromatic amino acids. The recombination was more difficult than anticipated, as we were unable to properly isolate the strain with successful homologous recombination. Instead the bacteria transformed with the oprF, which encodes a membrane protein that leads to improved extracellular shuttle-mediated electron transfer from the Bielefeld 2013 iGEM team, was tested against the G. sulfurreducens PCA wild type. The results showed that the nanowires in G. sulfurreducens were more durable and conductive than E. coli with OprF, thus making nanowires a better alternative for use in MFCs. Insertion of G. metallireducens pilA Into G. sulfurreducens Proved Difficult A fragment consisting of pilA from G. metallireducens, camR, and the 500 up- and downstream regions of the endogenous pilA genes of G. sulfurreducens were successfully constructed by PCR. The fragment was validated on gel electrophoresis, as seen in Figure 27-Left, with the band just above 3000 bp. The first electroporation was performed, and afterwards chloramphenicol was added for a concentration of 10µg/ml, this was added to isolate the G. sulfurreducens recombinants, as done by Coppi MV et al. 2001 Coppi MV, Leang C, Sandler SJ, Lovley DR. Development of a Genetic System for Geobacter sulfurreducens.
Applied and Environmental Microbiology. 2001;67(7):3180-7.. After the selection of the G. Sulfurreducens recombinants, a PCR of its genomic DNA was carried out to identify the expected 3000 bp insertion. However, we were only able to retrieve a 1700 bp fragment, as seen in Figure 27-Right, and not the expected 3000 bp fragment. For this reason, a new experiment using the same 3000 bp fragment was performed, this time increasing the chloramphenicol concentration to 30 µg/ml for the selection of recombinants. This, however, resulted in no bacterial growth after electroporation. Due to a lack of time, we eventually decided to put this part of our project on hold. Figure 27. Left: DNA fragment consisting of PilA gene with LacI promoter and RBS from G. metallireducens, CamR and the 500 bp up- and downstream regions to pilA on G. sulfurreducens. Right:The PCR product from G. sulfurreducens’ genome, which grew on a chloramphenicol concentration at 10µg/ml with primers 500 bp up- and downstream from pilA. Nanowires From G. sulfurreducens Have Higher Electrical Conductivity Than E. coli Even though we could not accomplish implementation of nanowires in E. coli, nor optimise the nanowires in G. sulfurreducens, we still found it relevant to test the G. sulfurreducens wild type nanowire efficiency, against previously used methods for extracellular electron transfer in MFCs in the iGEM competition. For that reason, we tested the electrical conductivity of G. sulfurreducens PCA wild type against E. coli ER25663127 containing OprF, a membrane protein, which helps carrying electrons to an extracellular electron shuttle in form of methylene blue. Figure 28. Single chamber, three electrode systems were used for measuring the electrochemical current generation. The measurements were done with an electrode potential at -0.2V(vs. Ag/AgCl KCl sat.). Starting from the left: E.coli with OprF, E.coli wild type, and then G. sulfurreducens wild type. The results achieved from this experiment is shown in Figure 29, which shows that by using nanowires as the electron carrier, it is possible to tremendously increase the electrical current compared to the extracellular electron shuttle used previously. As seen for the E. coli wild type, the current decreases over time, which indicates no activity. E. coli containing the membrane porin OprF shows a rapid increase, followed by a gradual decrease over 6-8 hours. The current of E. coli remains low, while G. sulfurreducens' current starts increasing after 20 hours, unfortunately the experiment had to stop due to time constraints. Figure 29. Illustration of the microbial current generated from G. sulfurreducens wild type, E. coli ER25663127 wild type, and E. coli ER25663127 containing OprF. The measurements was done with an electrode potential of -0.2V (vs. Ag/AgCl KCl sat.). Ten minutes before the measurements, IPTG and methylene blue were added to the E. coli containing chambers to achieve concentrations at respectively 1mM and 0.1mM. Acetate was added as electron donor to achieve a concentration of 10 mM to the G. sulfurreducens ten minutes before the measurements. This shows that the electrical current is limited by time for the E.coli with OprF, which Bielefeld also concluded. They measured the voltage over time, and their results showed that it reached the maximum after 30 mins. Highly interesting, the current from G.sulfurreducens starts to increase after 20 hours. This makes G.sulfurreducens a much more viable and stable electron transporter than the electron shuttle, OprF. Parts & Procedures In this section, you will find all the needed information to replicate our approach and experiments. The constructed parts, notebook, SOPs and protocols will show in a pop-up window from which you can obtain all the necessary knowledge, should you be interested. An essential part of going to the lab is risk and safety assessments, which you will find at the end of the section.
Week 8 Proper Risk Management
Biosafety and proper risk assessment are important aspects to consider before any handling of genetically modified organisms (GMOs). There are several concerns that must be addressed properly. The safety of the public as well as of the environment is of the utmost importance, but the safety of the person in direct contact with the GMOs should not be compromised either. The risk associated with laboratorial work can be evaluated using the statement “Risk = Hazard ✕ Probability”. To responsibly address this inquiry, the entire team was given a mandatory lab safety course held by Lab Technician Simon Rose. In addition, we received a detailed handbook regarding lab safety. This ensured that all our team members were well equipped to work safely in the lab. Throughout the project we have continuously been evaluating the safety of our work. These assessments can be found in the safety form. Furthermore, our team participated in the 5th annual BioBrick workshop hosted by DTU BioBuilders. Here we participated in a lab safety course before entering their laboratories. Both of these lab safety courses gave us the necessary knowledge to work safely with GMO and handle waste appropriate, as well as the according procedures in case of an emergency.
Public and Environmental Risk Assessment
The chassis organisms containing the system is meant to be contained in a device, which is incorporated into an urban environment. While this device would be a safely enclosed container, it still possess the risk of physical breakage from violent acts or environmental disturbances. For this reason, we consulted a plastics expert, who advised us to use the plastic known as Polycarbonate. This plastic is remarkably durable, with the ability to ward off most physical traumas. The plastics expert has estimated that such a container would last in an urban environment for at least 20 years, and most likely more than that. To illustrate the durability of the plastics, he notified us of several devices from the 1980s made of the same plastic, which still stand strong today.
Biodiversity
Biological diversity is defined as: The variability of living organism from all sources. Terrestrial and marine, and of cause the ecological complex they are a part of. This also include diversity within species, and between species and ecosystemsConvention on Biological Diversity.
List of Assessed Items Chassis Organisms Practices “Change is the law of life; and those who look only to the past or present are certain to miss the future” John F. Kennedy Welcome to our Human Practices! Now, when it comes to the particulars of our Human Practices, you will find that it has been separated into three main parts. This is all strictly for narrative purposes, as every single aspect of our project and Human Practices are deeply intertwined through a shared philosophy: If you want change, look to the future!
1. A Philosopher’s Guide to the Future To ensure an ethically sound iGEM product and experience, we have discussed the ethical considerations that ought to be taken into account. As luck would have it, one of our team members is a philosopher with an interest in bioethics. Thus a guidebook was created, a guide that amongst other things includes an overview of some of the bioethical arguments iGEM participants are likely to encounter, when discussing synthetic biology. You can find these considerations in our section on Bioethics. 2. An Implementation of the Future We reached out to our local Municipality of Odense along with various experts, for their advice on the development and implementation of our device in an urban environment. You can read more about this in our section on Integrated Human Practice. 3. A Trip to the Future and Beyond! Considering how our main philosophy was: If you want change, look to the future! It seemed prudent to bring our message on sustainability to the next generation. You can read more about our efforts in the section on Education and Public Engagement. “The facts of life... to make an alteration in the evolvement of an organic life system is fatal. A coding sequence cannot be revised once it's been established.” Tyrell, Bladerunner Synthetic biology and the iGEM competition are aimed to help solve societal issues, issues such as agriculture, medical research, and environmental resource management, the last of which has been our motivation throughout our project. However, while synthetic biology offers many new exciting possibilities, several concerns have to be met when dealing with living organisms. Against, the excitement of the iGEM community, skeptics have pointed to the uncertainty and potentiality for unwanted consequences that might arise from working with synthetic biology. We as a team decided to give these concerns some serious thought! We have had several conversations on topics such as meta ethics, applied ethics, sustainability, GMO, and so on. Furthermore, we established an open and honest mode of discourse to use when engaging with other teams and the public. We held a workshop in bioethics in relation to our Danish Meetup. This workshop consisted of a historical insight into the history of GMO provided by our very own historian, as well as a hefty, kind-hearted debate, supervised by our own philosopher.
“The best way to predict your future, is to create it” Abraham Lincoln - (former) president of the United States of America Not that we can claim to be anything like Abraham Lincoln, or even vampire hunters. Nonetheless, we do agree that to create the future we all hope for, we must contribute to find a sustainable solution for a greener future. Before we can tackle the task of providing a sustainable future for the entire world, we must first look to our own local environment. We believe that the best way to gain a better understanding of a global dilemma, is to examine how a local environment is affected by it. Hopefully, this approach will help future iGEM teams find a connection between global issues and local ones. This approach has helped us elucidate specific issues and find sustainable solutions, which can be implemented into our society with the help and endorsement of local agents. A Statement from the Mayor of Odense We first decided to reach out to the mayor of Odense, to investigate the possibilities for iGEM to help in the government's endeavours to make Odense a CO2 neutral city. “We face a series of challenges that we have to recognise, in the chase of the good and sustainable life in the city of Odense. Some of these concern local circumstances, while others contain national and even global issues. We as the municipality can only go so far on our own. So, we are entirely dependent on the help of local agents. It makes me so happy, when the students of the city, have taken on the mantle of developing new green technologies that solves global issues, while contributing to local city growth” Peter Rahbæk Juel - Mayor of Odense The core philosophy of our Integrated Human Practices has been to incorporate local experts in the development of our project. We wanted to examine how results produced in the laboratory, could be used to shape a product that corresponds with the green values of Odense. Interviewing Smart City Odense For the possible implementation of the PowerLeaf in the local environment of Odense, we decided to reach out to Kristina Dienhart, project manager of Smart City Odense. Smart City Odense is a project within Odense Municipality, that seeks to combine urban planning with new technologies and open-data toward creating a smarter, more sustainable city. Mrs. Dienhart introduced us to several considerations that shaped large parts of our project. We do not know the needs of every urban area in Odense and consequently, we have aimed to create a device that is changeable to a city in movement such as Odense.
Interviewing the City Renewal Project My Bolbro
Rikke Falgreen Mortensen is the manager of the Bolbro’s city renewal project called Mit Bolbro i.e. My Bolbro. We arranged a meeting with her with the intent of further investigating how the PowerLeaf could and should be integrated in an urban area of Odense, in this case the neighbourhood of Bolbro.
Bolbro is an old neighbourhood in Odense historically known to be the home of the working class. While Bolbro provides a homely atmosphere known to the locals, it has had a hard time attracting new residents. However, this is subject to change as the neighbourhood in 2016 received approximately 1.6 million US dollars to renew its city space and to create an even more appealing, and vibrant neighbourhood. This will be achieved by including the locals, as Bolbro is characterized by having a strong, engaging civil society. Mrs. Mortensen is not only an expert in urban renewal, but also in how to include local citizens in reshaping the public space in which they reside. We had a discussion with Mrs. Mortensen about the creation of a prototype based on the wishes of Bolbro’s local citizens. Following this conversation, she provided us with a pitch that aimed to help us develop this prototype. “Hauge’s square is a spot in Bolbro, which we aim to make a central place in Bolbro; a place that invites the citizen to meet and dwell. Your solution should be able to contribute to help citizens recharge their phones, e.g. a solution could be implanting the PowerLeaf into a interactive furniture, but where the demand an aesthetic pleasing design still remains.” - Translated from Danish “A part of the vision of this project is the concept of making a pop-up park with differently designed multi-furniture, preferably in wood and organic design, which are removable to the various areas where we are going to develop in the district. It is furniture that should be able to be used to relax in and at the same time also motivates children to move. There is also a need for charging devices and it therefore demands that your solution is an integrated, but still mobile solution, as the park will move physically over time. Finally, the playground is to be developed especially for the young audience, which is a major consumer of power for phones. The playground must be a place where youngsters hang out after school, while maintaining its status as a green space. - Translated from Danish” The making of the furniture as a prototype called for a revisit of our safety concerns. We now knew that children would be climbing and playing on the furniture, making it crucial that the material of the PowerLeaf will not break. This is a concern we discussed with Flemming Christiansen, which you can read all about next. Finding the Proper Material Criteria to the Prototype Interview with Flemming Christiansen For the purpose of finding the necessary materials for our prototype, we contacted one of the leading plastic experts in Denmark, Flemming Christiansen , who acts as the market development manager of SP Moulding. He has been acting as a plastics consultant since his graduation as a master of science in Engineering, with a speciality in plastics. A meeting was quickly arranged for the purpose of confirming our criteria, the technical design, the material, and the possible price of creating the PowerLeaf. In accordance with our established criteria, Mr. Christiansen suggested that we use the plastic known as Polycarbonate, specifically Lexon 103R-III Polycarbonate. Unfortunately, the material cannot fulfil the criteria on its own. He therefore suggested that we take a few liberties with it. In order to prevent UV degradation to the exposed parts, we will be adding certain additives to the surface. This increases the UV resistance of the device, without hindering the sunlight from reaching the bacteria.
Click here to see a detailed version of the prototype. Workshop with Business Developer Ann Zahle Andersen For the purpose of getting a business perspective on our project, we met with Business Developer Ann Zahle Andersen. She arranged two workshops for us based on a business model canvas. She encouraged us to view our project, as if we meant to make startup business. These workshops gave us a better comprehension of society’s pull and pushes on a project like ours. This forced us as a team to get to the bottom of what we found important about our project. Upcoming Meeting with Borgernes Hus Borgernes Hus i.e. House of the Citizens is a new initiative offered by the City Central Library. The initiative aims to offer guidance and advice to projects such as ours. It is meant to aid Odense in its journey towards the status of becoming a modern Danish city. Unfortunately, the building remains under construction until after our trip to Boston, meaning that they have been busy with the construction while our project was underway. It is for this reason that we, along with director Jens Winther Bang Petersen, agreed that a future collaboration would be the most suitable solution.
If you want change, look to the future! Such was the wording of our core philosophy. A philosophy that was carried out, by reaching out to the people of our society to ensure the engagement of the next generation, within the world of synthetic biology.
Danish Science Festival At the Danish Science Festival we hosted a workshop for kindergarteners, during which we taught them about synthetic biology, sustainability, the history of GMO, and bioethics. The children would in turn teach us as well, as they showed us the endless possibilities for bacteria designs, through the “Draw-a-Bacteria”-contest. This inspired us to reevaluate our initial idea. School Project Interview with 6th Graders Following the Danish Science Festival, we were contacted by two enthusiastic 6th graders, Bastian and Magnus. The two boys wanted to learn more about iGEM and GMO, which they intended to write about in a school project. They were curious to what range GMO could be used, and how we utilised it in our project, the PowerLeaf. UNF Summer Camp The UNF Summer Camp is an opportunity for high school students to show extra dedication towards science. We talked to some of the brightest young minds imaginable, all of whom aim to work in different fields of science in the future. At the summer camp, we held a presentation about our project, the iGEM competition, as well as how to handle and work with genes. We taught them how to assemble BioBricks and provided them with BioBricks for DNA assembly experiments, creating a ‘hands-on’ experience for these enthusiastic teenagers.
The Academy for Talented Youth We hosted a workshop for the Academy for Talented Youth, an association for some of the most talented high school students in Denmark. During the workshop we invited the students into our laboratories, where they conducted a miniprep and a gel electrophoresis on bacteria containing our BioBricks. Additionally, we held a presentation and discussion about our project, with the dedicated students. We strongly believe in mutual communication and made sure to compile feedback, all of which was positive! Presentations for the Local Schools The local high schools, Mulernes Legatskole and Odense Tekniske Gymnasium, invited us to present our project, in addition to starting discussions with the students about GMO. Final Presentation at SDU-Denmark The day before we travelled to Boston, we booked one of the big auditoriums at the University of Southern Denmark, for the final rehearsal of our jamboree presentation. We made sure to take note of all the feedback and tips we received, while also implementing these into our final presentation. This event was promoted on all the information screens at our university in order to attract a broad audience and increase the interest for iGEM. Thus, making it possible to reach a substantial amount of future applications for the SDU-iGEM team and ensure that the iGEM spirit will continue to prosper in the future! Social Media Social media is an easy way to impact a high number of people, so a strategy was concocted with the intention of reaching as many people as possible with our outreach.
Our strategy yielded marvellous results, amongst which was a video on our project, that reached viewers equal to 16% of our hometown’s population, along with becoming the second most seen bulletin of the year from University of Southern Denmark. They have also asked us to film our experiences at the Jamboree, which will feature on the homepage of the student’ initiative BetonTV.
Several articles were written about our project in local newspapers, one was even featured in the saturday special. Prospects The aim of our prospect section is to expand on the vision of the PowerLeaf; a vision we would love to see realised. An overview of the project has been created, in the hope that it will benefit future iGEM teams. Additionally, it is aimed to assist iGEM teams-to-be, should they wish to take the PowerLeaf to the next level.
Building a Product for a Better Future The purpose of the PowerLeaf is to provide a greener alternative to the currently available sources of energy. An important aspect of such an undertaking is to limit the use of depleting resources in the construction of the device itself. This is accomplished through the use of the most common resources available, namely recyclable plastics and bacteria. In turn, this will contribute to our dream of building a better future. A future where fear of reaching a critical shortage of natural resources has been eliminated. Another benefit of the PowerLeaf is the bacteria's ability to self-replicate, if provided with the essential nutrients.
Genetic Code Expansions for Biological Engineering In an effort to advance the current technologies used in synthetic biology, research groups are working on genetic code expansion. We had an interesting talk from postdoc and former iGEM participant, Julius Fredens, about his work on genetic code expansion. Once a technology like this expands, it will completely revolutionise biological engineering, including that of the PowerLeaf. Genetic code expansion could be used for optimisation of the systems in the PowerLeaf, such as optimisation of nanowires, improvement of the light-sensing system, and making the breakdown of cellulose inducible.
Hello future iGEM’er and congratulations on starting your iGEM journey! You are going to have an amazing time with plenty of wonderful experiences, new friendships, and an extensive amounts of obtained knowledge. In this section some ideas for improvement and further development of our project, the PowerLeaf, will be presented. Further Development of Our Project For those of you that are interested in our project and would like to improve upon it, this is the section you have been looking for. We have listed the systems and the related information needed for the fulfillment of the device we envisioned. Nonetheless, you should not feel restricted by these suggestions. You shall be more than welcome to contact any of us regarding questions about the project. You can find each of our team members contact informations in the Team section in the credits. Investigated and Researched Systems Subjects that Ought to be Implemented in the Device Credits “You'll stay with me?” - “Until the very end” J.K. Rowling, Harry Potter and the Deathly Hallows Just like in the movies, you only get to meet the brilliant minds behind the project in the closing credits. Some might leave the cinema without sitting through the credits, but we hope you will sit through ours, as you will get to know us on a more personal level. We probably have more in common than you think. And do not forget that behind every great team is an equally great amount of external attributions. The contributors have supported and inspired us, especially when things have been rough and deadlines closeby. When you finish this section, we kindly ask that you turn your attention to our various collaborations, all of which have been amazing experiences. They truly show of that wonderful iGEM spirit!
Welcome to the team page! Here you will get to know us on a more personal level. Our team is made up by 12 students from 8 very different majors. As friends, we experienced the most amazing summer together, filled with various fun activities, both in- and outside the lab. We would like to mention a few. We had road trips, dinners, Game of Thrones nights, heck, we even celebrated Christmas in July! All of this was shared with our amazing supervisors, to whom we are truly grateful.
Ellen Gammelmark Study: Biochemistry and Molecular Biology Emil Bøgh Hansen Study: Biology Emil Søndergaard Study: History Emil Vyff Jørgensen Study: Physics Felix Boel Pedersen Study: Biochemistry and Molecular Biology Frederik Bartholdy Flensmark Neergaard Study: Biochemistry and Molecular Biology Frederik Damsgaard Højsager Study: Medicine Jonas Borregaard Eriksen Study: Pharmacy Lene Vest Munk Thomsen Study: Philosophy Malte Skovsager Andersen Study: Biochemistry and Molecular Biology Sarah Hyllekvist Jørgensen Study: Biochemistry and Molecular Biology Sofie Mozart Mortensen Study: Biomedicine Project Synergy We have all been working together in every aspect of our project. Nevertheless, some people have had to focus on some areas more than others. The main groups are listed as follows; To ensure a good team spirit and dynamic we formulated a cooperation agreement.
"Alone we can do so little; together we can do so much" Helen Keller The American author Helen Keller had it right! As an iGEM team, you can reach many goals, but as an entire community, we can aspire to achieve so much more. We would like to thank all the people that made our iGEM experience so memorable, we truly enjoyed your companionship! Danish Ethics and Wiki Workshop at SDU In the spirit of the iGEM community, we hosted a meetup in August for our fellow Danish iGEM teams: InCell from the University of Copenhagen (KU), and the Snakebite Detectives from the Technical University of Denmark (DTU). A total of seven members from these two teams joined us for breakfast and attended our meetup. This was the first ever iGEM meetup hosted by our university, so we decided to make it memorable. We took advantage of our interdisciplinary team roster, and thus designed a wiki and ethics workshop to aid our fellow Danish teams.
Attending Meetups Besides hosting our own meetup, we also attended several meetups during our iGEM experience. The first of which, was the 5th Annual Biobrick Workshop in March, hosted by the Technical University of Denmark, DTU-Denmark. This meetup not only gave us our first experience with Biobricks, but also worked as a foundation for friendships across different teams.
Further Collaboration In regard to our project, we have been in contact with the iGEM teams from Bielefeld and Imperial College, who helped us by sending crucial parts, relevant to the execution of our project.
Nothing can be done alone, so please scroll further to read about the contributors, who helped make this project a reality. Laboratory, Technical and General support We would like to give a special thanks to our supervisors: We would also like to thank: Sponsors Thanks to: Litterature Thank you for your time! We hope you enjoyed our wiki and getting to know our project.
PowerLeaf - A Bacterial Solar Battery
Abstract
Our adventure began with a meeting between strangers from eight different studies. Despite our different backgrounds, we had one thing in common; a shared interest in synthetic biology. Soon after this first meeting, we were herded off to a weekend in a cottage - far away from our regular lives. The cottage was a place to bond and discuss project ideas. It immediately became apparent that being an interdisciplinary team was going to be our strength as each member had unique qualities that enabled them to efficiently tackle different aspects of the iGEM competition. So, we made it our goal to take advantage of these qualities.
We decided to make a proof-of-concept project. Specifically, we wanted to use bacteria as a novel and greener solution for solar energy storage. This project was later dubbed the PowerLeaf – A Bacterial Solar Battery.
Since it is a one-page wiki, you can just keep on scrolling, and you will be taken on a journey through our iGEM experience.
Achievements
Meet all the Deliverables Requirements – You are reading the team wiki now, so that is one cat in the bag. You can find all attributions made to the project in the Credits section of the wiki. The team poster and team presentation are ready to be presented at the Giant Jamboree. We also filled the safety form, the judging form and all our parts were registered and submitted.
Clearly State the Attributions – All attributions made to our project have been clearly credited in the Credits section.
Improve and/or Characterise an Existing Biobrick Part or Device – The characterisation of the OmpR-regulated promoter BBa_R0082 was improved, as the level of noise was studied on different vectors.
Induction and inhibition of the pBAD promoter, BBa_I0500, were studied, whereby the characterisation of this part was improved.
Furthermore, we characterized if the periplasmic beta-glucosidase could make E. coli live on cellobiose in fluid medium BBa_K523014, submitted by the 2011 iGEM Edinburgh Team.
The data obtained in these experiments are presented in the demonstration and results section.
Collaboration – We have collaborated with several teams throughout our project by taking part in discussions, meetups, and answering questionnaires - we even hosted our first meetup for our fellow Danish iGEM teams. You can read all about this in the Credits section.
Human Practices – Our philosopher, historian, and biologist have discussed the ethical and educational aspects of our project in great detail. In extension to their work, we have been working extensively with education and public engagement .
Model Your Project – Through extensive modelling, we have learned that it is possible to regulate bacterial dormancy. However, the modelling showed that it would be inadequate to only regulate the toxin RelE, as this would make the bacteria unable to exit dormancy. To regulate dormancy properly, would also require tight regulation of the antitoxin RelB. This information was used to shape the entire approach of the light-dependent dormancy system.
World Situation
Fortunately, we learned through interaction with local agents that a great deal of people share our belief: that we ought to pursue the development of low energy cities with a high quality of life. In fact, we even discovered that our own hometown Odense wants to be the greenest, most renewable city in Denmark by 2050 Odense Municipality’s website, regarding their politics on the current climate changes..
In the pursuit of this goal we rose to the challenge of creating a truly green solution, which would provide an environmental friendly source of energy.
Please keep scrolling if you wish to read more about our solution, or go straight to bioethics if you are curious why we not only could, but ought to do something about the current and forthcoming energy crisis.
Our Solution
The device was originally designed to resemble a plant leaf aimed to provide a nature-in-city ambience. This hypothetical implementation of the PowerLeaf in an urban environment was developed through careful consideration, public engagement, and collaborations. We worked with local city planners from our hometown Odense, as well as with a plastic specialist from SP Moulding, the purpose of which was to advance our pre-established design, as well as attaining other changeable designs.
Our vision was clear and ambitions were high. As it turned out though, we had aimed too high, considering the limited timeframe, so at an early stage, we decided to focus on the following features:
Project Design
Dormancy System
When not exposed to light, PCB activates the phytochrome Cph1, thus promoting kinase activity through the EnvZ kinase. When the transcription factor OmpR is phosphorylated by EnvZ, expression of genes regulated by the OmpR-regulated promoter is initiated. Excitation of PCB by red light results in a situation where the transcription factor OmpR is not regulated. The absence of phosphorylated OmpR leads to no activation of the OmpR-regulated promoter, thereby preventing gene expression.
You can read more about the modelling here.
The model showed that the system is sensitive to the RelE:RelB ratio, as well as the total amount of produced toxin. As seen in Figure 2, implementation with production rates in the vicinity of 50 and 35 molecules per minute for RelB and RelE respectively, was found to be suitable for balancing our system; the bacteria lay dormant within the computed time and re-enter an active state within minutes.
The simulated data made it evident that implementing an optimised dormancy system comprises a challenge, as the individual expression levels of RelE and RelB, as well as their interaction, has a crucial impact on the regulation of dormancy. Thus, controlled gene expression of both RelE and RelB is required to implement a controllable dormancy system in the PowerLeaf.
If you want to dig deeper into this crucial modelling of the dormancy system, read the full results here.
On basis of the modulated system, the potential of different vectors and promoters in various combinations was tested. This constitutes the foundation for how the design of the light-dependent dormancy system in E. coli has been optimised, and the final approach shaped. Ultimately, the light-dependent dormancy system, which is illustrated in Figure 3, was composed of the following parts:
Light Sensing System
The genes needed for inducing dormancy when the bacteria are not exposed to light, are found in the photocontrol device part, BBa_K519030. This part is composed of three genes named ho1, pcyA, and cph8, all of which are essential to ensure the cells ability to respond to red light. When the photocontrol device is exposed to light, a phosphorylation cascade activates the transcription factor OmpR, which in turn induces transcription through the OmpR-regulated promoter, BBa_R0082. This system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. In the first considered design of the light-dependent dormancy system, the aim was to clone the photocontrol device, BBa_K519030, under control of a constitutive promoter, RelE under control of the OmpR-regulated promoter, BBa_R0082, and RelB under control of a constitutive promoter, all into one high copy BioBrick assembly plasmid pSB1C3, as seen on Figure 1.
From the constitutive promoter family the weak promoter, BBa_J23114, the two medium promoters, BBa_J23106, and BBa_J23110, and the strong promoter, BBa_J23102, were tested by fluorescence microscopy to determine which one to use for expression of the photocontrol device and RelB. Overnight cultures of the submitted parts expressed in E. coli TOP10 illustrated a clear gradient of increasing red fluorescent protein (RFP) expression correlated with the strength of the promoter, as seen on Figure 2.
Regulation of the OmpR-dependent Promoter Required a Low Copy Vector
The first construct containing the genes required for the light-induced dormancy was designed as shown in Figure 1. As the conducted modelling clarified, the necessity for stringent regulation of the RelE and RelB expression, the properties of the OmpR-regulated promoter were studied thoroughly. To assess the functionality of the OmpR-regulated promoter in practice, a reporter system containing the OmpR-regulated promoter controlling RFP was cloned into the E. coli strain MG1655 ΔompR. The phenotype of the resulting cultures revealed a dysregulation of the OmpR-regulated promoter. Thorough research lead to the finding that the OmpR-dependent promoter is not controllable when cloned on a high copy vector. As the modelling revealed, and which is evident from Figure 2-Main-Page, a relatively low expression of RelE is required to induce dormancy, whereas high expression levels quickly result in overshooting. Since the OmpR-regulated promoter is an integrated part of the light sensing system, replacement is not an option. Therefore, the variability of the relE gene copy number was studied, and it was found that the OmpR-regulated promoter should be cloned into the bacterial chromosome or a low copy vector to obtain proper regulation Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2.. This intriguing finding let to the aspiration to investigate the controllability of the OmpR-dependent promoter on vectors with different copy numbers compared to the chromosome, thereby improving the characterisation of the promoter for the benefit to future iGEM teams.
To incorporate DNA onto the bacterial chromosome, homologous recombination with the red λ recombinase is a suitable approach Thompson JF, de Vargas LM, Skinner SE, Landy A. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination. Journal of molecular biology. 1987;195(3):481-93.. Using this technique, a short fragment of chromosomal DNA at the bacterial attachment site attB Groth AC, Calos MP. Phage integrases: biology and applications. Journal of molecular biology. 2004;335(3):667-78. can be replaced with a linear DNA fragment encoding the OmpR-dependent promoter, RelE, and an chloramphenicol resistance cassette. Using polymerase chain reaction (PCR), the linear DNA sequence was flanked by sequences, which are homologous to part of the chromosome. The linear DNA fragment was electroporated into bacteria containing the pKD46 plasmid, encoding the red λ recombinase Thompson JF, de Vargas LM, Skinner SE, Landy A. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination. Journal of molecular biology. 1987;195(3):481-93., which mediated the recombination. The fundamental concept of this approach is illustrated in Figure 3.
An Inducible Promoter was Chosen to Regulate the Gene Expression of the Antitoxin RelB
Originally, it was thought to place the antitoxin RelB under a constitutive promoter of appropriate strength. However, as the modelling revealed, strict regulation of RelB is essential to counteract the toxic effect of RelE and enable a functional dormancy system. Thus, it was deduced that it would be more suitable to utilise an inducible promoter for this purpose and it was decided to put RelB under control of the LacI-regulated, lambda pL hybrid promoter, BBa_R0011. The William and Mary team found that the LacI-regulated, lambda pL hybrid promoter had a low level of noise, a measure of the variability in gene expression between cells in the population, when cloned into a low copy vector. Therefore, this regulated promoter was chosen.
To mimic the expression of RelB, a reporter system composed of the LacI-regulated, lambda pL hybrid promoter and GFP was assembled. Hereby, the appropriate concentration of IPTG required to induce the promoter on a low copy vector, was identified. During the cloning, it was discovered that the site between the LacI-regulated, lambda hybrid promoter and this particular construct under the tested conditions had formed a hotspot for transposons. In the light of this finding, another inducible promoter was chosen. The HKUST-Rice iGEM 2015 team demonstrated that induction of the AraC-regulated promoter, pBAD, caused a gradual increase in gene expression when cloned into the low to medium copy plasmid, pSB3K3, in contrast to an all-or-none behavior when cloned into the high copy vector, pSB1K3. Taking these results into consideration, the pBAD promoter was used in combination with the pSB3K3 vector to regulate the expression of the antitoxin RelB. Again, a reporter system was used to mimic the expression of RelB, as the part, BBa_I6058, containing YFP controlled by pBAD, was assessed on different vectors.
The Final Approach for the Three Components Comprised Three Different Vectors
Based on the modelling the system approaches reviewed in the preceding part, the final design, which is illustrated on Figure 4, was established. Ultimately, the dormancy system was composed of the photocontrol device controlled by the PenI-regulated promoter on a high copy vector, RelB controlled by pBAD, BBa_K2449031, on a low copy vector and RelE controlled by the OmpR-regulated promoter on either a low copy vector or the chromosome.
Carbon Fixation
Cellulose Biosynthesis
The ability for G. xylinus to produce cellulose nanofibers from UDP-glucose, crystallize, and secrete it, is controlled by genes in the Acetobacter cellulose synthase (acs) operon acsABCD. This operon encodes four different proteins: AcsA, AcsB, AcsC and AcsD. A dimer, known as AcsAB, is formed by a catalytic domain, AcsA, and a regulatory domain, AcsB. This dimer is responsible for synthesising the cellulose nanofibers from UDP-glucose, whereas AcsC and AcsD secrete cellulose and form an interconnected cellulose pellicle around the cells Mehta K, et al. Characterization of an acsD disruption mutant provides additional evidence for the hierarchical cell-directed self-assembly of cellulose in Gluconacetobacter xylinus. Cellulose. 2014;22:119–137., as illustrated in Figure 6.
Breakdown of Cellulose
Cellulose is a long polysaccharide consisting of β-1,4-linked D-glucose units and many organisms, including E. coli, lack the enzymes able to degrade these strong β-linkages. To overcome this, the C. fimi has developed two cellulases, namely the endo-β-1,4-glucanase and exo-β-1,4-glucanase, respectively encoded by the cenA and cex genes Jung
SK, Parisutham V, Jeong SH, Lee SK. Heterologous Expression of Plant Cell Wall
Degrading Enzymes for Effective Production of Cellulosic Biofuels. Journal of Biomedicine and Biotechnology. 2012;2012.. The endoglucanase is able to randomly degrade the amorphous structure of cellulose, thereby allowing the exoglucanase to cleave the β-1,4-linkages at every other D-glucose unit. Thus, disaccharides are released in the form of cellobiose Lam TL, Wong RS, Wong WK.
Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in
Escherichia coli by the reduction of promoter strength. Enzyme and microbial
technology. 1997;20(7):482-8., as illustrated in Figure 7. Cellulose itself is too large to be transported across the bacterial cell membrane, and therefore, the breakdown of cellulose into cellobiose must take place in the extracellular fluid.
The ɑ-hemolysin transport system is an ABC transporter complex consisting of three proteins, namely the outer membrane protein TolC, hemolysin B (HlyB), and hemolysin D (HlyD) Gentschev I, Dietrich G, Goebel W. The E. coli
alpha-hemolysin secretion system and its use in vaccine development. Trends in
microbiology. 2002;10(1):39-45., which can effectively transport intracellular hemolysin A (HlyA) to the extracellular fluid. Utilising a linker peptide, the protein of interest can be fused with HlyA. Once a protein is HlyA-tagged, it can be recognised by the ATP-binding cassette HlyB, which will initiate transportation of the HlyA-tagged protein to the extracellular fluid, as seen in Figure 8 Gentschev I, Dietrich G, Goebel W. The E. coli
alpha-hemolysin secretion system and its use in vaccine development. Trends in
microbiology. 2002;10(1):39-45. Su
L, Chen S, Yi L, Woodard RW, Chen J, Wu J. Extracellular overexpression of
recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in
E. coli BL21(DE3). Microbial Cell Factories. 2012;11:8..
While cellulose is too large to be pass the cell membrane, transportation of cellobiose is a common feature found in many organisms. An example is E. coli that utilises the membrane protein lactose permease (LacY) Sekar R, Shin HD, Chen R. Engineering Escherichia coli Cells for Cellobiose Assimilation through a
Phosphorolytic Mechanism. Applied and Environmental Microbiology.
2012;78(5):1611-4., whereby the cellobiose is enzymatically catabolised in the cytosol.
Through evolutionary events, many organisms have developed the ability to express enzymes, capable of breaking the β-linkage in cellobiose. E. coli expresses the periplasmic β-glucosidase encoded by the bglX gene, which is known to have said feature, hydrolysing the cellobiose β-linkageUniProt entry for bglX. Saccharophagus degradans expresses a different enzyme that efficiently cleaves the β-linkage in cellobiose, namely cellobiose phosphorylase encoded by the cep94A gene. This enzyme phosphorylates the cellobiose at its β-linkage, resulting in the degradation of cellobiose to D-glucose and α-D-glucose-1-phosphate Sekar R, Shin HD, Chen R. Engineering Escherichia coli Cells for Cellobiose Assimilation through a
Phosphorolytic Mechanism. Applied and Environmental Microbiology.
2012;78(5):1611-4., as seen in Figure 9.
In the endeavour to engineer E. coli to utilise cellulose as its only carbon source, inspiration was drawn from the Edinburgh 2008 iGEM team project, who developed two BioBricks containing the cenA and cex genes. In this project, the α-hemolysin transport system was utilised by creating HlyA-tagged endo- and exo-β-1,4-glucanases, using a peptide linker. To implement this system in E. coli, heterogeneous expression of hlyB, hlyD, cenA-hlyA and cex-hlyA was required.
To achieve this, DNA synthesis of cenA and cex was ordered, each tagged with HlyA. The genes encoding HlyB and HlyD were retrieved from the part BBa_K1166002 by phusion PCR. Using the resulting PCR product, the following construct was composed for the degradation of cellulose into cellobiose, as illustrated in Figure 10.
The Edinburgh 2011 iGEM team created a BioBrick with the bglX gene, which is endogenous to E. coli, in the endeavour to increase the efficiency of the degradation of cellobiose to glucose. However, it seems that the enzymatic activity of the periplasmic β-glucosidase has faded as a result of evolution, rendering E. coli incapable of surviving solely on cellobiose. Thus, even though E. coli can absorp cellobiose, it is not able to survive with this as its only carbon source.
As a solution to this, a part containing the cep94A gene was synthesised, with the intend to enable E. coli to survive solely on cellobiose. Thus, a construct containing cep94A controlled by a LacI-regulated promoter was composed, as illustrated in Figure 11.
Extracellular Electron Transfer
Electrochemical devices, such as batteries and fuel cells, are broadly used in electronics to convert chemical energy into electrical energy. A Microbial Fuel Cell (MFC) is an open system electrochemical device, consisting of two chambers; an anode chamber and a cathode chamber, which are separated by a proton exchange membrane, as illustrated in Figure 12. Both the anode and the cathode in an MFC can use various forms of graphite as base material and in the anode chamber, microbes are utilised as catalysts to convert organic matter into metabolic products, protons, and electrons Khanal YLaSK. Microbial Fuel Cells, Capter 19. In: Khanal. EbYLaSK, editor. Bioenergy: Principles and Applications. First Edition ed: Published 2017 by John Wiley & Sons, Inc.; 2016.. This is carried out through metabolic pathways such as glycolysis, thereby generating ATP needed to maintain cellular life. This metabolic pathway also releases electrons, which are carried by NAD+ in its reduced form, NADH.
Nanowires are long electrically conductive pili found on the surface of various microorganisms, such as the metal reducing Geobacter sulfurreducens. G. sulfurreducens utilises nanowires to transfer accumulating electrons retrieved from metabolism, to metals in the nearby environment Mahadevan R, Bond DR, Butler JE, Esteve-Nuñez A, Coppi MV, Palsson BO, et al. Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling. Applied and Environmental Microbiology. 2006;72(2):1558-68.. This gram-negative bacteria is strictly anaerobic, as it is unable to transfer its electrons to the environment in the presence of the highly reducing oxygen. Nanowires found in G. sulfurreducens are type IV pili polymer chains composed of pilA monomers, and they can reach a length of nearly 10 mm Richter LV, Sandler SJ, Weis RM. Two Isoforms of Geobacter sulfurreducens pilA Have Distinct Roles in Pilus Biogenesis, Cytochrome Localization, Extracellular Electron Transfer, and Biofilm Formation. Journal of Bacteriology. 2012;194(10):2551-63.. The proteins required for the effective transfer of electrons by nanowires is a complex and poorly understood system, which includes an extensive series of c-type cytochromes, as shown in Figure 14 Morgado L, Fernandes AP, Dantas JM, Silva MA, Salgueiro CA. On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochemical Society transactions. 2012;40(6):1295-301..
It was then decided to work on optimisation of the G. sulfurreducens by increasing the electrical conductivity of its endogenous nanowires. To achieve this, synthesis of the pilA genes from G. metallireducens was ordered, which was used to create a BioBrick. Using the same approach for homologous recombination as in the dormancy system, a DNA fragment containing the chloramphenicol resistance cassette of the pSB1C3 vector, was made for later selection of recombinant G. sulfurreducens. The PCR product was ligated with fragments retrieved from the 500 bp upstream and downstream regions of the chromosomal pilA genes of the G. sulfurreducens PCA strain, creating the fragment seen in Figure 15.
Demonstration & Results
Dormancy System
To assess which of the constitutive promoters would be suitable for a uniform expression of the photocontrol device, BBa_K519030, and the antitoxin RelB, BBa_K2449028, the expression levels and the noise of four different members of the Anderson promoter collection and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, BBa_J23114, BBa_J23110, BBa_J23106, and BBa_J23102.
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:
As the strong constitutive promoter exhibited the most uniform expression, this was chosen to regulate the expression of the photocontrol device genes. With the modelling results in mind, it was decided that the relB gene should be regulated by a tightly controllable uniform promoter, thereby ruling out the constitutive promoter family members as a possibility.
The cloning of the strong constitutive promoter, BBa_J23102, and the photocontrol device, BBa_K519030, emerged problematic. For further information about these difficulties, read here.
Consequently, the applicability of two different promoters was studied. These were the PenI-regulated, BBa_R0074, and the Mnt-regulated promoters, BBa_R0073, whose repressors are not found in E. coli, making the gene expression constitutive in this organism.
The relative expression and noise levels were quantitatively assessed using fluorescence microscopy of YFP reporter systems, BBa_I6103 and BBa_I6104, expressed on pSB1C3 in MG1655 at OD600=0.3-0.5. For this purpose, an Olympus IX83 with a photometrics prime camera was used, set to an excitation at 470 nm, emission at 515-560 nm, and exposure time at 20 ms.
Proper regulation of the OmpR-dependent promoter, BBa_R0082, is necessary for the implementation of a functional dormancy system, as the balance between RelE and RelB is imperative. To verify that the OmpR-regulated promoter is up to the task, a reporter system containing RFP under control of the OmpR-regulated promoter, BBa_M30011, was cloned into E. coli strain SØ928 ΔompR, lacking the OmpR transcription factor, on a high copy vector. By using a ΔompR strain, the background generated by stimulation of the intrinsic OmpR system is removed, and the strain functions as a negative control.
RFP expression was assessed by fluorescence microscopy using an Olympus IX83 with a photometrics prime camera, set to an excitation at 550 nm, emission at 573-613 nm, and exposure time at 500 ms. Assessing the RFP expression by fluorescence microscopy, it was discovered that the OmpR-regulated promoter mediated gene expression even in the absence of its transcription factor, see Figure 17. This observation was confirmed by going through the literatureLevskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2..
All things considered, the OmpR-regulated promoter was found to exhibit leaky expression comparable to the expression levels mediated by the constitutive promoters. When cloned into a low copy vector, the leaky expression was reduced prominently. Thus, to obtain proper regulation of RelE expression by the OmpR-dependent promoter, a low copy vector is required.
To control the gene expression of RelB, the LacI-regulated lambda pL hybrid promoter, BBa_R0011, was chosen. Due to a putative formation of a transposon hotspot between the promoter sequence and the GFP reporter, which is described further here, another inducible promoter was chosen to regulate the expression of RelB.
The pBAD promoter holds great potential to regulate the expression of the relB gene in our system, as it is capable of both an induction and repression. The HKUST-Rice iGEM team from 2015 found that the pBAD promoter exhibits an almost all-or-none behaviour upon induction with arabinose when located on a high copy vector, but allows for gradual induction when cloned into a low copy vector. Thus, it was evident that this promoter on a high copy vector would be inappropriate for the regulation of RelB expression. Based on these findings, a low copy vector was used to investigate the ability to inhibit gene expression subsequent to induction of pBAD.
RelB expression was simulated by fluorescence microscopy using a pBAD-YFP reporter system, BBa_I6058.
For this purpose, an Olympus IX83 with a photometrics prime camera was used, set to an excitation at 470 nm, emission at 515-560 nm, and exposure time at 150 ms. Transformed E. coli MG1655 cells were cultured in M9 minimal medium supplemented with 0.2% glycerol and 30 µg/mL chloramphenicol, to avoid catabolite repression from glucose residues present in LB medium. Two cultures were incubated, of which one was induced with 0.2 % arabinose from the beginning. At OD600=0.1, designated time 0, the cultures were split in two and 0.2 % glucose was added to one of each pair. Samples were obtained at time 0, before division of the cultures, and at 30 min, 60 min, and 120 min.
The resulting images revealed, that the inducer arabinose was required to stimulate expression of YFP, and that the addition of the repressor glucose to a uninduced culture had no effect. Furthermore, it was evident that addition of arabinose induced expression of YFP, and that subsequent addition of glucose terminated the pBAD regulated gene expression on a low copy vector, resulting in a reduced fluorescence level. 30 minutes after inhibition this reduction was already evident, and after 120 minutes the gene expression controlled by pBAD was even further decreased, as seen in Figure 19.
Determination of Noise Levels
The images obtained by fluorescence microscopy, which are given in Figure 1, revealed that the weak promoter, BBa_J23114, presented high variability in the expression of RFP between cells, indicating a high level of noise. Contrarily, RFP expression was more uniform in the bacterial cells containing the medium promoter Bba_J23106. Similarly, another medium strength promoter BBa_J23110, showed uniform level of RFP expression, although less consistent than the observed for BBa_J23106. Expectedly, the strong promoter, BBa_J23102 exhibited high RFP expression in cells and a low level of noise was observed. 
Comparing these findings to the fluorescence microscopy data, it was hypothesised, that the increasing noise could be caused by bacterial plasmid loss. This could be ascribed to degradation of ampicillin in the medium, since the mechanism behind ampicillin resistance relies on the β-lactamase mediated cleavage of the antibiotic, thereby relieving the selective pressure. Consequently, the bacteria, which have lost their plasmid could be able to compete with the bacteria still containing their plasmid.
The fluorescence microscopy for the strong promoter did not exhibit a considerable level of noise, however, this does not reject the hypothesis. The increase in noise was revealed by flow cytometry, as measurements were carried out at several times throughout growth. However, the fluorescence microscopy measurements were only carried out once in the exponential phase, where the noise was not prevalent yet.
To substantiate the hypothesis, the experiment was performed anew using M9 minimal medium supplemented with 0.2 % glycerol and 100 µg/mL ampicillin, in an attempt to decrease the plasmid loss rate and optimise the selection.
The resulting data revealed, that the decrease in fluorescence levels had a lower rate for both promoters when the bacteria were cultured in M9 minimal medium, seen in the right graph in Figure 2, than in LB medium, seen in the left graph in Figure 2. Furthermore, when the bacteria were grown in M9 minimal medium with double amount of antibiotics compared to the LB medium, they displayed a higher level of fluorescence. Some of these observations could potentially be ascribed to the increased selection, that was strived after in this experiment. Moreover, the difference between the measured fluorescence levels for the two reporter systems was notably lower. This could indicate, that the reason behind the difference in expression levels between the weak and strong constitutive promoters, is due to the fact that bacteria cloned with the weak constitutive promoter are more prone to plasmid loss. However, it was observed that the bacteria expressing RFP under control of the weak promoter, did in fact fluoresce with a lower intensity.
To verify the hypothesis regarding the plasmid loss, the cultures were plated on 50 µg/mL ampicillin LA selection plates as well as LA plates without antibiotic, 12 hours after incubation start. For comparison, cultures containing either chloramphenicol and kanamycin resistance cassettes, were likewise plated out after similar treatment. The average plasmid loss measured as non-resistant CFU as a percentage of the total population is seen Figure 3.
Transposon Hotspot
Breakdown of Cellulose
The results showed that cep94A, which expresses cellobiose phosphorylase, was functioning as expected. However, we were unable to detect any traces of the desired endoglucanase (cenA) and exoglucanase (cex) secreted by the type I secretion system.
Extracellular Electron Transfer
Parts
Notebook
Notebook
As 12 strangers we met for the first time on the 22nd of February. We cooked and ate a lovely meal together, and hereby had our first team building experience.
Week 10
We went to the DTU-Denmark BioBrick tutorial event, where we had an unforgettable experience. Here, we met some of the other Scandinavian teams, with whom we had a wonderful time.
Week 11
A group contract was discussed and the first version was written.
As part of the promotion of the team and iGEM, a banner was created for the information screens at our university, the University of Southern Denmark.
Week 12
Excursion to a cabin on Langeland. At this idyllic spot, different ideas for our project were discussed and narrowed down to only a handful of ideas. It was a fun weekend and friendships among the team members began to sprout.
Week 14
Through an iterative process, the list of project ideas was cut down. Ultimately, the PowerLeaf - A Bacterial Solar Battery was chosen as our project.
Week 16
The team had a weekend of lab training with the supervisor Patrick, where we learned about all the basic applications for cloning. The work on the dormancy system had begun!
Week 17
We had a lab safety course with Lab Technician Simon Rose. Thereby, we became qualified for unsupervised laboratory work. Now we were properly equipped to start the work!
During the Danish Science Festival, we presented our iGEM team. Here, we talked to a lot of people about GMO, especially with children. We had prepared several activities for the children to participate in, one being a drawing competition. In this competition, the most innovative and creative idea of a bacteria won. From this activity, the team received a lot of fantastic drawings, with endless creative ideas, that the team took into consideration and used to further shape our project.
Week 18
The team had a course in innovation with Ann Zahle Andersen, from the patent office of the University of Southern Denmark. Here, we gained a better understanding of our project from a business point of view.
Bastian and Magnus, 6. grade students from a local public elementary school, interviewed us on the topic of GMO. After the interview, the boys received a guided tour around the iGEM lab. The boys used the obtained information about GMO to write an assignment at their school.
Week 19
We got the first look at promoter reporter systems in the fluorescence microscope.
Week 21
The team started their collaboration with Odense Municipality. The main focus in this collaboration was to integrate our project into an urban setting.
Week 23
Putting our final touches on the PowerPoint presentation for the Nordic Meetup, held by the KU iGEM team. This was the first time, our project was presented for other teams. At the meetup, the team members met a lot of wonderful people, some for the first time, others were enjoyable reunions.
Week 24
We received parts from the Bielefeld team for the carbon fixation system. There was a breakthrough regarding the secretion system, as we succeeded in the creation of the cex part.
Week 26
The team filled out the first three parts of the safety form. We had a talk with postdoc Oona Snoeyenbos-West, who suggested that we should work with Geobacter sulfurreducens to test the bacterial nanowires.
Week 27
The entire team went to the Netherlands, where we joined the European Meetup at TU Delft. At this meetup, we had the pleasure of meeting new people from several European iGEM teams, alongside meeting old friends from the Nordic teams. The team extended the trip while in the Netherlands, to spend a day in beautiful Amsterdam together.
Week 28
The genes encoding RelE and RelB, which comprise a toxin-antitoxin system, were purified from E. coli.
Week 29
We cracked the code of modelling and our modelling system stabilised. Furthermore, the secretion system was created.
Week 30
A presentation was held at the UNF (Danish Youth Association of Science) high school camp, where we taught them about synthetic biology and how to work with genes.
In the lab, we received parts from the Imperial College team for cellulose biosynthesis. We also began creating the BioBrick for our nanowires. The cenA part was created, and the assembly of the entire secretion system was begun.
Week 31
Our lab workers analysed the properties of the LacI-regulated, lambda pL hybrid promoter for the energy storing unit.
Week 33
The team celebrated an early christmas. Yes, in July! Crazy, we know! There was a christmas tree, a cosy atmosphere and more food, especially gravy, than should be possible to consume. The team had a jolly good time.
We finally made our PCR fragment for Geobacter, which would be used for homologous recombination. Testing on our cellobiose phosphorylase, that we received from IDT, was begun.
Week 34
We held our own SDU meetup with focus on wiki and ethics for KU and DTU. The reunion with the other Danish iGEM teams was a welcoming experience.
Week 35
The abstract for the iGEM Jamboree was finished. In this week we also decided that we should enter the energy track.
Week 36
The team members working on human practices had a really informative meeting with Kristina Dienhart from Smart City Odense.
We held a presentation at the local high school OTG (The Technical High School of Odense). SDU communication helped us produce a video about our project, which reached more than 27000 people on Facebook. Furthermore, an article about our project and team was published in the local newspaper Fyens Stiftstidende.
The genes encoding the carboxysome were assembled. Furthermore, the expression levels of the Mnt- and PenI-regulated promoters were compared in the fluorescence microscope.
Week 37
We hosted an event for ATU (Academy for Talented Youths) with transformation and synthetic biology as the main focus. This began with a general presentation, after which we went to our lab, where the aspiring scientists from ATU performed plasmid purification and gel electrophoresis. The team had a marvelous day as it was wonderful to see so many young individuals being engaged in science.
Week 38
We had a human practises meeting with Rikke Falgreen Mortensen about integrating our project in MyBolbro.
Furthermore, we held a presentation about GMO and our project for the local public school, Odense Friskole.
We were surprised to discover that a transposon hotspot had formed between the LacI-regulated, lambda pL hybrid promoter and the GFP reporter.
Week 39
We held a presentation at the local high school, Mulernes Gymnasium, where synthetic biology was the main focus.
The lab workers discovered that a region containing the strong constitutive promoter and the BioBrick prefix had vanished from the photocontrol device plasmid. Furthermore, it was decided to keep the cellulose biosynthesis subproject theoretical henceforth.
Week 40
Materials for our prototype were set after a talk with the plastic expert Flemming Christiansen.
Second part of the course in innovation and patents with Ann Zhale Andersen was completed.
The last two part of the safety form part were completed and submitted. Additionally, it was decided to keep the carbon fixation subproject theoretical henceforth.
Week 41
The final promoter for the RelB expression, pBAD, was chosen and cloned.
We ceased trying to perform the recombination in Geobacter after several difficulties.
Week 42
We got together with the old iGEM teams from SDU-Denmark at the Old-iGEM Meetup. Both the project in general and our wiki were presented to them, and we got a lot of useful feedback to improve our project, presentation, and wiki. We shared a lovely meal and had a great night.
We managed to create our secretion system part after several tries and problems.
Week 43
Parts were sent to HQ and the Judging form was filled out.
In the lab, we obtained promising results, as the features of the pBAD and OmpR-regulated promoters were studied in the fluorescence microscope and flow cytometer.
Besides working in the lab to get the last results ready for the wiki freeze, the team spend the weekend going through all of the text.
Week 44
With the wiki freeze around the corner, we worked day and night, caffeine concentrations in our blood running high. We shall make it!
SOPs and Protocols
Safety
In the lab, we worked with several potentially harmful chemical agents such as dimethylsulfoxide (DMSO), ethidium bromide, chloroform, phenol, Congo red, antibiotics, and autoclaved glycerol. These chemical agents were handled using gloves at all times, and whenever deemed necessary, handled in a fume hood. Gloves were worn when necessary, and clean lab coats were worn restrictedly in the laboratorial areas. To visualise bands in agarose gels, we used an UV board. UV rays are carcinogenic when exposure is frequent and prolonged. To reduce the amount of exposure, several precautions were made. Gloves, long sleeves and a facial screen were worn at all times, and the time spend at the UV board was kept at a minimum.
One of the biggest concerns would be the release of GMOs into nature. While the GMOs used are not pathogenetic, they would be able to share the plasmids containing antibiotic resistance selectors to other bacteria, that might be pathogenic. Antibiotic resistance in pathogenic bacteria complicates the treatment of an infected individual and could, in tragic cases, be the difference between life and death. However small the risk of this scenario might be, it should be addressed properly. Furthermore, antibiotic resistant E. Coli strains could outmatch some of their fellow E. Coli strains through natural selection. This could negatively affect the balance of nature, that we are aiming to restore with the development of the PowerLeaf.
To avoid these risks, several kill switch mechanisms should be implemented into the final device. This could be performed by implementation of a kill switch activated by exposure to light in the energy converting unit. This would of course mean, that the energy converting unit’s container would need to block all sunlight. A task that could easily be carried out by adding Carbon Black to the required areas of the container. The energy storing unit, which requires light to function, could then have a kill switch which makes it dependent on the presence of the energy converting unit. This could be accomplished by having harmless molecules, not naturally found in nature but required for the survival of the energy converting unit, circulating in the system. A similar effect could be accomplished by making the bacteria in the energy converting and storing units codependent on each other for their survival. The implementation of such kill switch mechanisms would tremendously improve the biosafety of the device by opposing hazards related to any kind of physical breakage.
From a biologist point of view, this include all genes, species and ecosystems of a region.
Biodiversity is threatened by climate change. The climate change have not caused extinctions, but have lead to a decrease of fitness in a number of species. By reinventing how to use transgenic organisms, the aspiration is to improve on the degrading biodiversity that can already be seen. If you want to learn more about the impact caused by GMO and climate change on biodiversity you can read more here.
Escherichia coli strains: K12, TOP10, MG1655, KG22, BW25113, DF25663127, SØ928
Geobacter Sulfurreducens strain: PCA
Vectors
pSB1A2: An iGEM plasmid backbone carrying a ampicillin resistance gene
pSB1A3: An iGEM plasmid backbone carrying an ampicillin resistance gene
pSB1C3: An iGEM plasmid backbone carrying a chloramphenicol resistance gene
pSB3C5: An iGEM plasmid backbone carrying a chloramphenicol resistance gene
pSB1K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB4K5: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB3K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
Bacteriophages
P1 phage, using its site-specific recombinase for transduction of E. Coli
Bioethics
- A Philosopher’s Guide to the Future
All of these considerations, debates, and results led our philosopher Lene to write a longer guidebook entailing an overview of the various bioethical arguments often made for and against the use of synthetic biology. The guidebook is mainly an insight into the most important ethical considerations made by our team, e.g. how they shaped our product, but it also offers a personal and clear overview of arguments and principles meant to help future teams to get a conversation on ethics started!
Make sure to give this guidebook a read, it is definitely worth it!Integrated Practices
- An Implementation of the Future
We sought the advice of experts in other fields, e.g. expert in plastics to design the best possible exterior of our device. Furthermore, we met with business developer Ann Zahle Andersen to investigate the core value of our product from a business perspective.
We believe that Human Practices have played an essential role in our iGEM project. Everything from the design of our prototype to ethical considerations have been influenced by the people we engaged with.
This made us aware of necessities essential to Odense and its citizens. She gave us feedback that we integrated into numerous areas of our overall project.
Furthermore, this interview was also a source of inspiration for our ethical and safety thoughts. While we ought to strive for a sustainable tomorrow, we do not necessarily have to provide an exhaustive description of what the future should look like.
Mrs. Mortensen also argued that a changeable design would be the optimal solution to fit the challenges one faces in creating a vibrant, green city ambience. Such a task depends on different preferences, laws and needs. A technology needs to be both flexible and accessible to successfully contribute to the process of creating an engaging city environment. She showed great interest in our device and even offered to implement it in the parks of Bolbro, should the product become a reality.
The system itself will consist of two different compartments, an outer and an inner chamber. The first will be facing the sun, while the other will be facing the building or furniture. Since one culture of the bacteria depends on solar energy to produce its product, the outer compartment must allow for sunlight to pass. It should here be noted that the prototype is purely hypothetical, as the membrane, between the two compartments, should exclusively be permeable to cellulose. For that reason, we wanted to find a material, that fulfilled our established criteria, so that we could illustrate the technology.
The device itself will be made entirely from plastic, a material that is thought to be undesirable due to the difficulties in its disposal. This is due to plastic being of a xenobiotic nature, making it generally recalcitrant to microbial degradation Fewson CA. Biodegradation of xenobiotic and other persistent compounds: the causes of recalcitrance. Cell. 1988.. Following these concepts, we can identify the following set of criteria for the desired material:
During our conversations with Mr. Christiansen, we reached the topic of what to do in case of a breach. Should the container against all expectations be damaged, the environment will be exposed to the GMO inside. The solution we came up with was the possible implementation of a kill-switch in the inner compartment, making it vulnerable to sunlight. Should the bacteria of said unit be exposed to sunlight, they would perish. As the outer compartment would be dependent on the continued coexistence of the two units, the entire GMO system would be purged in case of a breach . To implement this feature, the inner chamber would be covered with Carbon Black, which has the ability to absorb sunlight, thus leaving the compartment itself in darkness.
The process of constructing our device would be through an extensive use of Injection Moulding, which is considered pricey equipment. The material is expensive at 4-5.5 USD per kg at orders above 1 metric ton, according to Mr. Christiansen, but its longevity and durability means it would not need to be replaced for a long time. Lastly, we discussed the reusability of Polycarbonate, which he assured us was of no concern, as the material could be reused and recycled with ease.
It is our hope, that a collaboration with Borgernes Hus will be of assistance to future iGEM teams from SDU as well as other students from Odense.
Education & Public Engagement
- A Trip to the Future and Beyond!
Ever since World War II, the West has seen an expansion and intensification of anti-scientific sentiment, which today primarily concern Genetically Modified Organisms (GMO). We will for that reason explore GMO’s role in history, to see if a historical perspective will allow us reach a new understanding of these sentiments. You can read all about it here.
One of the high school students suggested that the Powerleaf should be able to rotate according to the sun, to ensure maximum exposure and outcome. We took this brilliant advice into consideration and contacted Robot Systems Engineer student, Oliver Klinggaard, who helped us with the potential implementation of a pan/tilt system. He provided us with his recent project report on the subject, as well as a description of the adjustments required for the implementation in our system, which you can find here.
Two students from the UNF Summer Camp thought the PowerLeaf was an interesting approach to sustainable energy, and they wanted to hear even more! So, they contacted us in late October, as they were interested to work on a project about green technology.
An 8th grade class from the local public school, Odense Friskole, were invited to see our laboratory workspace. It was a challenge to successfully convey our project and the concept of synthetic biology in a way that would be easily understandable by 8th graders, who have only recently been introduced to science. A challenge that we accepted and solved, by relaying the fundamentals in synthetic biology, e.g. the basics of a cell, DNA, and GMO.
From all of these presentations and interactions with younger individuals, we had a strong intuition that it had made an influence on their awareness of synthetic biology. This intuition was supported by the positive feedback provided by teachers and students. An awareness of how new scientific technologies can be a feasible solution to a possible energy crisis. Technologies such as synthetic biology, with endless capabilities to achieve efficacy, since no one knows what tomorrow brings. For more information about this read To Future iGEM Teams
You can read all about our social media strategy and results here. The commercial can be seen right here:
Perspectives
As tools for genomic editing improves, the advancement of biological devices will conceivably become even more complex and independent. They will do so by introducing new metabolic pathways originated from other organisms using genetic engineering. This could potentially allow the PowerLeaf to become completely independent through its self-replication. Independency would occur, when the bacteria are modified to produce their own essential nutrients directly from unwanted pollution in the environment. This trait would lead to cleaner cities, along with providing a natural solution of sustainable energy.
To Future iGEM Teams
Utilised by the energy storing unit, the dormancy system can reduce metabolism during times of the day with no solar energy available, e.g. at nighttime. We encountered a few challenges during the assembly and optimisation of the dormancy system. However, through modeling we obtained vital knowledge on how to regulate the system. If you are interested, you can read more about the work performed on the light-dependent dormancy system.
We received the required parts from the Bielefeld 2014 iGEM team and began assembling the parts into one fully functional BioBrick.
However, we encountered some trouble cloning these parts. You can give it a go anyways, or maybe even try to redo the carbon fixation by using a system from a different organism. Regrettably, we eventually had to let go of this system, so we could focus on other components of the PowerLeaf. If you are interested, you can read more about the work we did regarding the carbon fixation.
These parts were retrieved from the 2014 Imperial College iGEM team. Assembly of these long BioBricks emerged to be troublesome, which regularly occurs when cloning long sequences of DNA. Thus, this part could be the very thing to improve, as it is a rather central part of the system. If you are interested, you can read more about our work on the cellulose biosynthesis and secretion.
Employed by the energy converting unit, this system degrades cellulose to glucose, from which electrons could be retrieved. After extensive work the system showed promising results as our bacteria were able to grow on cellobiose. If you are interested, you can read more about the work we did regarding the cellulases.
Inspired by a previous study Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, et al. Expressing the Geobacter metallireducens pilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity. mBio. 2017;8(1)., we set out to create the required BioBricks for the system. However, to successfully implement optimized nanowire in G. sulfurreducens, further work would have to be performed. If you are interested, you can find additional information about the work we did regarding the nanowires.
Harvest of solar energy comes to mind as one of the most essential systems needed for the PowerLeaf to actually work. We had to select parts of the PowerLeaf to work on, and at the end of the day, we decided to cut this subpart. Instead, we had a great Skype call early on in the process with the Australian Macquarie iGEM team, who has been working for several years with the implementation of the photosynthetic systems in E. coli.
This is also a very crucial part of the PowerLeaf, since it would otherwise be generating an electrical current non-stop, even when not needed. Thereby overthrowing the potential for long term-storage of solar energy. We believe this can be solved either through precise gene circuit regulation or by physical compartmentalisation, but there might be even more elegant ways in which to handle this challenge.
It should be possible for the energy storing unit to convert CO2 into cellulose, thereby producing O2 making its chamber aerobic. For the energy converting unit to effectively transfer retrieved electrons to an anode, it requires anaerobic conditions. Thus the removal of O2 from the device is an obstacle to overcome. It will require some out-of-the-box thinking to come up with a novel idea without having to require more energy than produced.
To make a workable prototype of the PowerLeaf, proper engineering of the hardware is necessary. This includes elements such as anodes, chambers, circulation of important nutrients, removal of wastes, and the use of appropriate materials. We worked out the optimal type of plastic for the system with the help of a local expert. You can read about our work regarding the plastic here.
Finally, we do not want you to miss out on the ‘after-the-credits-clip’ that summarises the fun we had during this fantastic iGEM adventure. This is will be the moment you will get that long-awaited ‘thank you for listening, we hope you enjoyed our wiki and project’.
Team
E-mail: elgam15@student.sdu.dk
Why, hello there! My name is Ellen and I have spend most of my waking hours either in the lab with a pipette in hand, or just outside the lab with a computer on my lap. You know - learn iGEM, live iGEM, love iGEM!
E-mail: ehans15@student.sdu.dk
Howdy! I’m the first of many Emil’s and the team's only biologist! I am a huge wolf enthusiast! This summer I left my boots in the closet, in order to put on a proper lab coat and suit up for iGEM. In addition to my time in the lab, I have also looked into how GMO can influence the environment and what we need to do to ensure a safe iGEM project.
E-mail: emsoe09@student.sdu.dk
Ahoy thar! My name is Emil, and I want to be the next Indiana Jones. But before I can raid any tombs, I’ve decided to raid iGEM trophies. When I’m not cooking or travelling, I’m drawing on my background in history for communications and human practices.
E-mail: ejoer15@student.sdu.dk
Mojn! I am yet another Emil. I might not be a model biochemist, so instead I am modelling biochemistry! My iGEM existence is a stochastic binary function between naps and extreme bursts of energy.
E-mail: feped15@student.sdu.dk
Aloha. My name is Felix and I bring joy to others by eating my daily ryebread with paté and wearing my magical red racer rain coat. Speaking of magic, I’m the team’s wiki lizard (get it?). I also do dry-lab, and whenever I miss the “sunlight”, I kindly join the others in the wet lab.
E-mail: frnee15@student.sdu.dk
Hey yo! I’m Frederik and I have worked day and night on iGEM, mostly drinking beers at night time, but that should count as well. When I’m not working in lab or on the PC, I have fun with my teammates and tell bad dad jokes. I also make crazy ideas come true - like celebrating Christmas in July.
E-mail: frhoe14@student.sdu.dk
Heyah! I’m the other Frederik. I’m a green, lean, coffee-machine. I’ve been the steady supplier and consumer of coffee on the team. My main focus has been on how to build a sustainable iGEM project. I’ve been planting trees, eating green and lowering our team's carbon footprint. Oh, and did I also mention that I starred in our commercial? You can get autographs later.
E-mail: jerik15@student.sdu.dk
Hey sup? I’m Jonas and used to like sports, partying, eating cake, hanging out with friends and other such things most people like to do. During iGEM these interests have changed… As I have been enslaved in the lab, I’ve come to realise that the only purpose of my life is to be in the lab.
E-mail: letho11@student.sdu.dk
Hey, is it solipsistic in here, or is it just me? When not wondering whether or not there is an external world, I’ve been busy working out how to implement our solar battery into our local community and what to gain from doing so. Oh, and imposing metaethics on my team members, but I Kant go into detail with this here.
E-mail: malta14@student.sdu.dk
Eyy, I’m Malte. I’ve mostly been working in the lab wrapped in the dankest of lab coats, doing the most exciting of experiments. All in the name of why the heck not. In the lab the utmost highest level of patience is needed, especially when tasked with testing if BioBricks function as intended. This has, as seen in the image, sadly caused me to pull out most of my hair.
E-mail: sajo415@student.sdu.dk
Despite my favorite occupation is digging into literature, my main attribution to our project has been to run around in the lab. Luckily, there is a clear link between wet- and dry-lab. Even though I am the smallest member of the SDU iGEM team, I have definitely risen to the occasion. iGEM has been an amazing period of my life!
E-mail: sofmo15@student.sdu.dk
Hi there! My name is Sofie, and I am the team mama! I am the one who makes sure everyone gets their fair share of cake and baked goodies. When I’m not in the kitchen busy making cakes for my teammates, you can find me in the lab with a pipette in my hand.
Nothing can be done alone, so please scroll further down, to read about all the people who contributed to making our project successful. We are so grateful for all the help and support we have been offered throughout our iGEM experience.
Collaboration
We utilised the broad interdisciplinary profile of our team, to have Emil S. and Lene present the perception of science throughout the history and the meta-bioethical aspects of GMO, respectively. Emil S. has a Bachelor of Arts in History, and Lene has a Bachelor of Arts in Philosophy. The ethical presentation was purposely turned into an ethical debate, where viewpoints of ethical conduct were exchanged and discussed. After the presentations and discussions on metaethics, it was time for the wiki workshop.
The SDU-Denmark iGEM teams have won the Best Wiki prize several times in the past. As such, we wanted to share the knowledge gained from our university's experience. To facilitate this exchange of knowledge on wiki development, we recruited our current supervisor Thøger Jensen Krogh, to facilitate presentations on how to design a good wiki page. Thøger was qualified for this task through his role as the designer of the SDU iGEM 2013 and 2014 team wikis, both of which won the special prize on both occasions. During the presentation, Thøger had arranged several exercises where the attendees got to mingle, discuss and evaluate their wikis. This resulted in a steady flow of information and constructive feedback between all three teams.
After a long day of learning and discussing, we went for a tour around campus under the summer sun. This concluded in a visit to the roof terrace of the campus dormitory, followed by dinner. It was requested, by our fellow Danish teams, to make the SDU meetup an iGEM tradition.
Our second meetup, the Nordic iGEM Conference was hosted by the University of Copenhagen, UCopenhagen, and took place in June. The main focus of this meetup was the traditional mini Jamboree. Participating in this meetup gave us useful feedback from both the judges as well as from our fellow iGEM teams. This helped us greatly to shape and develop our project for the better.
To celebrate the beginning of our iGEM summer, we went on a road trip to attend the European Meetup, hosted by the Delft University of Technology in the Netherlands. Here we discussed ideas regarding our project at a poster session, and learned about all the other great iGEM projects. We also made new friends from all over Europe.
As our project revolves around global warming and green sustainable energy, we were thrilled to hear about the iGEM Goes Green initiative made by the TU Dresden iGEM team. Following their guidelines, we have calculated the carbon footprint of our laboratory work and travelling. We have, in part, tried to make up for our carbon footprint, by changing our travelling and eating habits in our everyday lives. Furthermore, we have reduced our daily electricity consumption, our wiki became CO2 neutral, and we made an effort to sort our waste. The full report can be scrutinised here. Due to our team being the most green dream team, TU-Dresden asked us to lead the iGEM Goes Green project in year 2018.
We sought expertise from the Macquarie Australia iGEM team, who has worked with the implementation of photosynthesis in E. coli since 2013. We had an interesting Skype call with their team, where we discussed the particular challenges their previous teams had experienced throughout their projects.
We were also able to help the Stony Brook iGEM team by facilitating communication with members of the SDU iGEM team from 2016.
During our project we received several questionnaires from fellow iGEM teams. We were delighted to help the teams by answering their questionnaires. These included from:
Attributions
Final Words
Now you can sit back, relax, and be proud of your hard work. While you do so, feel free to enjoy some of the less serious pictures and snippets from our amazing iGEM adventure.
