Team:SDU-Denmark/test
editors highlights
ENERGY STORED IN CELLULOSE • LIGHT-DEPENDANT DORMANCY SYSTEM • OPTIMISED NANOWIRES The PowerLeaf introduces a novel solution for long-term storage of solar energy, thus becoming an alternative to solar cells. This is accomplished without the use of environmentally harmful resources. The device is 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 public engagement and collaboration. The bacterial solar battery is composed of an energy storing unit (a) and an energy converting unit (b). The energy storing unit (a) is defined by a genetically engineered Escherichia coli that fixates carbon dioxide into the chemically stable polymer cellulose. A light-dependant system activates dormancy during nighttime to reduce energy lost by metabolism. The energy converting unit (b) uses genetically engineered Escherichia coli to consume the stored cellulose. Retrieved electrons are transferred by optimised nanowires to an anode resulting in an electrical current.
About Our Wiki Functionality on the Wiki Hi there, and welcome to our iGEM experience. I’m the creator of this wiki, and this year I decided to make it a one-page wiki - to make it stand out from the crowd. All you have to do is keep scrolling, and you will be guided through the project. You will always be able to follow your process through the wiki, by looking at the navigation bar. Whenever you want, you can click on one of the tabs, to get an overview of the sections associated with it. If you know where you want to go, you can click the section-links in each of the tab menus, and they will get you there immediately.
A Green Wiki Our project is all about ensuring a greener and more sustainable future for ourselves and the coming generations. This of course meant, that our wiki had to to follow this pursue. CO2NeutralWebsite sponsored our wiki with a CO2 quota equal to the amount of CO2 produced, by having the wiki running until 31-Oct-2018. This does not mean the wiki is CO2 neutral, but that the quote equal to its pollution is bought. Buying a quota means, that other companies won’t be able to buy this CO2 quota, thus, forcing them to improve their environmental policies if they wish to become CO2 neutral.
Introduction Welcome to our wiki! We are the IGEM team from the University of Southern Denmark. We have been waiting with great anticipation for the chance to tell you our story.
Bronze Medal Requirements 4/4 Register and attend – Our team applied 2017-03-30 and got accepted 2017-05-04. We had an amazing summer and are looking forward to attend the Giant Jamboree! Silver Medal Requirements 3/3 Validated part/contribution – Pending Gold Medal Requirements 3/4 Integrated Human Practices – Regarding the A Global Problem redo the approach in this section
In a Local Environment We are a team of young adults raised to be aware of climate changes and the potential limitations to the continuation of our way of life. We are a generation that appreciates open source and shared information. A generation that has 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, 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 would come to our booth with their parents to learn about bacteria, GMO, ethics and iGEM. After which, they would attend our “Draw-a-Bacteria”-competition. While drawing their own unique bacteria, they would present us with detailed stories about their design. See a selection of their amazing drawings here. Through this, we felt inspired and decided to revise our ideas. They even inspired the physical design for our original prototype; the PowerLeaf.
Our Solution The bacterial solar battery we envision, is composed of an energy storing- and an energy converting unit. The energy storing unit is defined by a genetically engineered Escherichia coli (E. coli). The E. coli uses solar energy for ATP production to fixate carbon dioxide into the chemically stable polymer cellulose, which essentially is the battery. A light sensing system activates dormancy during nighttime, in order to reduce energy lost by metabolism. The energy converting unit uses genetically engineered E. coli to consume the stored cellulose, by using an inducible switch. Retrieved electrons are transferred by optimised nanowires to an anode, resulting in an electrical current. 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 prospective iGEM teams to continue on the development of the PowerLeaf. We would love to see our project become a reality one day hence we have created a special page for future iGEM teams. This page includes suggestions for further development of the project.
Project & Results We have throughout the project worked on the development of 2 units for our device, an energy storing and an energy converting unit. Each of the systems we worked on for the units can be seen here: Light-dependant dormancy system Carbon fixation Cellulose biosynthesis and secretion Energy converting (G. Sulfurreducens) Breakdown of cellulose Extracellular electron transfer Once you reach each of the 5 systems in the 'Project Design'-section, you will first be given a short introduction to the underlying theory, which you will be able to expand on, by pressing “read more”. Which in turn will open a pop-up window with the additional information. After the theory, you will be given the approach used in each of the respective systems for the project. Before continuing on to the next system. To make things easier on you, we have developed icons to each of the above systems which will be used throughout the rest of the wiki.
overview picture, fix later, in a hurry, soft wiki Theory 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 makes up the two-component system naturally found in E. coli. The photoreceptor from phytochrome known as Cph1 was isolated from the cyanobacteria Synechocytis PCC6803. Cph1 has functional combination sites, which combined with the kinase EnvZ forms a two-domain receptor, known as Cph8. Activation of Cph8 is mediated by the 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..
Using the photocontrol device to control a toxin-antitoxin system is a system composed of two gene products, of which one specifies a cell toxin and the other an antitoxin, which neutralizes 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 neutralizes the toxic effect of RelE through interaction between the two proteins. Whether the cell lie 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 theory behind the light-dependent dormancy system, read here.
Approach
In 2004 the Austen and UCSF iGEM team created a device sensitive to light in regard to the Coliroid project. In this project, this system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. As the RelE-RelB system requires tight regulation 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., different systems have been duly considered in the endeavours to find a way to induce RelE without completely paralysing the cells. By modelling the toxin-antitoxin system and testing different approaches, a suitable and hypothetical working system-design has been implemented.
For further information about our approach, read here. Approach 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. This system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. In the first envisaged 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 #.
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 #.
On the basis of the data obtained by fluorescence microscopy, the strong constitutive promoter, BBa_J23102, was chosen to control the photocontrol device. For so far 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.
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 without success. In consideration of the fact that RelE is a bacterial toxin, the cloning of this gene was inconvenient. To account for this, overexpression of the antitoxin RelB is required. 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.
Controllable dormancy is a feature that holds the potential to be applied in many different situations. Inducing dormancy and bringing the bacteria back to a metabolic active state is like balancing on a tightrope. To establish the basis of the future implementations, the properties of this system would have to be investigated further. In an endeavour to provide this, mathematical modeling was performed in an attempt to prognosticate the system and simulate the interactions between the toxin and antitoxin. To consolidate the model, the capacity of the toxin-antitoxin system was assessed in an experiment. By manually regulating the RelB expression, the controllability of the dormancy system was studied.
Modelling
In order to find the best way to implement the RelE-RelB toxin-antitoxin system, modelling of the system was performed. The gillespie algorithm was utilised to model the interactions of the toxin and antitoxin. The toxin RelE is inhibited by the antitoxin RelB through complex formation, and both interact with their promoter in a feedback mechanism.
Theory
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. 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.. 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, which 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 theory behind 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 CO2 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 pairs of composite parts was achieved, however, the cloning of these parts with a promoter emerged 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.
Theory
Cellulose is a biopolymer produced by different species of gram-negative bacteria, especially by Acetobactors. An efficient producer of bacterial cellulose is Glucoacetobater xylinus, which produces large quantities of high quality cellulose. Cellulose is produced from the recourse glucose-6-phosphate. Glucose-6-phosphate is a key intermediate in the core carbon metabolism of bacteria given its importance in glycolysis, gluconeogenesis and pentose phosphate pathway. Even though the conversion of glucose and glucose-6-phosphate into cellulose is rather short, not only does the cell spends energy on forming UDP-glucose for cellulose production, it also uses glucose, which otherwise would have contributed to generation of ATP.
Approach
After some research it was found that cellulose would be the perfect source of energy between the two bacteria in our project…. Imperial College London made the project Aqualose (link) in 2014, during which they produced excess amounts of bacterial cellulose.
Theory
Cellulose is a natural polycarbonate used in nature for a vast variety of biological purposes. It is most commonly found in plants, where it serves as the main structural component. Since plants are primary producers, it has caused many organisms of the Earth’s ecosystem to adapt 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 Cellumonas fimi, which will convert 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
The α-Hemolysin Transport System
Uptake of Cellobiose
Breakdown of Cellobiose to Glucose Approach
Cellulose to Cellobiose
Cellulose to Cellobiose Theory
Microbial Fuel Cell
Bacterial Nanowires 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 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). did an exchange like this by heterogeneously expressing PilA from G. metallireducens, which proved to increase the electrical conductivity of the G. sulfurreducens recombinant by a 5000-fold. This optimisation can be helpful in the development of highly efficient bacterial strains for MFCs. With the intention of optimising a 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)..
Approach
Originally, we wanted to implement bacterial nanowires from G. sulfurreducens into E. coli. Through extensive research, we came to a similar conclusion as the Bielefeld 2013 iGEM team did; that this task was too comprehensive to undertake in the limited time of an iGEM project. Postdoc Oona Snoeyenbos-West suggested us to use G. sulfurreducens as the model organism for our MFC.
Parts & Procedures In this section, you will find all the needed information to replicate our approach and experiments. Both the parts, notebook, SOPs and protocols will show in a pop-window, from which you can obtain all the needed information, should you be interested. An essential part of going to the lab is risk and safety assessments. This you will find at the end of the section, so just go ahead and keep on scrolling.
Notebook SOPs Protocols 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 properly addressed. 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 shouldn’t be compromised either. The risk associated with laboratorial work can be evaluated using the statement “Risk = Hazard x Probability” (link). To responsibly assess 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 at all times. Throughout the project we have continuously been evaluating the safety of our work. These assessments can be found in the safety form (link).
Public and Environmental Risk Assessment
The chassis organisms containing the system is meant to be contained within a container, which should be 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. It is for this reason, that 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. As such the plastics expert believe that the 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 1970ies made of the same plastic, that still stand strong today.
List of Assessed Items Chassis Organisms Practices Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nam consequat sodales nisl at blandit. Suspendisse nisl tortor, dignissim vel ultricies ut, tincidunt eget nisi. Proin nec viverra erat. Vivamus commodo metus neque, non feugiat dolor viverra vel. Nullam sit amet elit luctus, interdum nisl fringilla, vestibulum libero. Nullam iaculis, purus non imperdiet vulputate, mi augue gravida lacus, eu sollicitudin lacus orci a ipsum. Aenean maximus porttitor viverra. Praesent sed fringilla mauris. Duis eu molestie orci, id pellentesque lorem. Interdum et malesuada fames ac ante ipsum primis in faucibus. Sed orci elit, sodales vel nibh sed, rhoncus ultrices dolor.
Jonas can approve on this “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 to be vampire hunters, but we do agree that to create the future we all hope for, we must contribute to finding a sustainable solution for a greener future. However, before we can tackle the arduous task of providing a sustainable future for the entire world, we must first look to our own local environment to better understand its vision for the future. Hopefully this approach will help future iGEM teams find a connection between global issues and local ones - as 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, and how it could possibly be tackled in such a setting. This approach has helped us elucidate specific issues and to find sustainable solutions that 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, with a high quality of life. “We face a series of challenges that we have to recognize, in the chase of a 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 dependant 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, which global issues while also contributing to the city's high quality of life.” Peter Rahbæk Juel - Mayor of Odense The core philosophy of our integrated human practices has been to integrate local experts in the development of our project; in order to better comprehend how to use the knowledge gained in the laboratory to shape a product that would compliment Odenses (or beyond) green values. We have also made use of experts in other fields in order to better understand how to shape our project - and so our human practice has influenced everything from the design of our prototype(s), laboratory work to ethical considerations.
Meeting with Kristina Dienhart For the purpose of a possible implementation of the PowerLeaf into the different areas of Odense city’s renewal, we decided to reach out to Kristina Dienhart. Kristina Dienhart was at this point in time project manager of Smart City Odense – a project within Odense Municipality that seeks to combine urban planning with new technologies and open-data, in order to create a smarter city. We decided to consult mrs. Dienhart, as Smart City Odense shares our core values; working transparently, openly and collaborative, while also sharing know-how. Mrs. Dienhart made us aware of the following necessities for Odense and its citizens - feedback that we have integrated in numerous areas of our overall project. It is important to note that at the time of our meeting with Mrs. Dienhart, our vision of the PowerLeaf was exclusively in the shape of a leaf; a leaf designed to be implemented on various buildings around Odense. Overall Mrs. Dienhart introduced us to several considerations that shaped large parts of our project. Her call for ‘the changeability aspect’ of the PowerLeaf has been used to reconsider the construction of the solar battery’s exterior and sustainability. We do not know the needs of every urban area in Odense; and consequently - with Mrs. Dienhart in mind – we have aimed to create a device that is changeable to a city in movement such as Odense. Mrs. Dienhart therefore challenged what we thought we wanted from a prototype - namely a fixed design - into the belief that we ought to create a prototype that can be shaped and reshaped depending on the requested necessities of the customer.
Meeting with Rikke Falgreen Mortensen
Mrs. Dienhart also helped to establish contact with Rikke Falgreen Mortensen, manager of the Bolbro’s city-renewal project called MyBolbro. We arranged a meeting with Mrs. Mortensen with the intent of further investigating how the PowerLeaf could and should be integrated into an urban area of Odense - in this case the neighbourhood Bolbro.
“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. At the same time it must also be an orientations point, from where citizens and visitors can find their way to other places and attractions in Bolbro. Today the possibility for enjoying the outside consists of the space in Hauge’s square, which is made up by a bakery, a small local library, and a parking lot. However, we believe that the space contains better opportunities. In short, the space must be transformed from primarily being a parking spot to a recreational place with a much more aesthetic design. Your solution should be able to contribute to help citizens recharge their phones, ex. A solution could be implanting the PowerLeaf into a ‘living’ furniture, but where demands for the aesthetic design still remains” “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 - and there should also be platforms that invite to activity ex. table tennis or a more screened seating for lovers, conversation or work. 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 church / playground is to be developed especially for the young audience, which is a major consumer of power for phones. The place must be a place where the youngsters hang out after school, still a green space where the solution should be integrated into the interior and could keep the target audience children and adolescents. The site is in a socially charged area, so it demands a robustness from of the solution, to help when faced with ex. vandalism” The making of the furniture as a prototype called for a re-visit of our safety concerns, as children will be climbing and playing on the furniture, it is crucial that the material of the PowerLeaf will not break; a concern we discussed with Flemming Christiansen, which you will be able to read more about next. Just keep scrolling! Flemming Christiansen Criteria to the Prototype Interview with Flemming Christiansen Meeting with Ann Zahle Andersen During our iGEM experience we met with Business Developer Ann Zahle Andersen twice. Mrs. Andersen had arranged two workshops for us based on a business canvas. This helped us to understand our project in a larger perspective. She encouraged us to view our project as if it was supposed to be a startup business, and through this perspective we gained a better comprehension of society’s pull and pushes on a project like ours. In a time of crisis she discussed our project’s advantages and disadvantages from a business perspective. A perspective and talk that forced us as a team to get to the bottom of what we found important about our project. And to truly appreciate the advice we have been given throughout our human practice work, as if we were a business trying to understand the needs of a costumer. Upcoming Meeting with Borgernes Hus ‘Borgernes Hus’ is a new initiative offered by the city’s central library. The name translates to ‘House of the Citizen’s’. The house aims to offer guidance and advice to projects such as our own. It is meant to aid Odense in its journey towards the status of a modern, danish city. Unfortunately, the building remains under construction until after our trip to Boston, meaning that they have been busy finishing said construction while our project was underway. It is for this reason, that we along with director Jens Winther Bang Petersen decided that a future collaboration would be the most suitable solution.
lets goo Prospects Our prospects section is aimed to expand on our visions regarding the PowerLeaf. A vision we would very much love to see become a reality. For this reason, we have concentrated on creating an overview of the project, for the benefit of future iGEM teams. Hopefully it can assist prospective teams on how to take the PowerLeaf to the next level. Lastly, we decided to list some of our project ideas to the teams wishing to create a completely new project. Perhaps some of these ideas can be used by prospective iGEM teams, or just help to kickstart their creative thinking.
Building a Product for a Better Future
The purpose of the PowerLeaf, is to provide a greener alternative to the currently available energy sources. An important aspect of such an undertaking, is to limit the use of depleting resources, such as silicon, in the construction of the device itself. This is accomplished through the use of the most common resources available. This will contribute to our dream of building a better future, where fear of reaching a critical shortage of natural resources has been eliminated. The production of the PowerLeaf itself is made easier too, as the device benefits from the bacteria's ability to self-replicate, if provided with essential nutrients.
Genetic Code Expansions for Biological Engineering Expanding beyond those technologies used in today's Synthetic Biology, many research groups are working on genetic code expansion. We had an interesting talk from post.doc Julius Fredens, about his work on genetic code expansion. Once a technology like this advances, it will completely revolutionize biological engineering, including that of the PowerLeaf. Genetic code expansion could be used for optimization of the systems in the PowerLeaf; optimization of nanowires, improvement of the light-sensing system and making the breakdown of cellulose inducible.
Hello future iGEM’er and welcome to the section where you are the center of attention. First of all, congratulations on starting your iGEM journey, you are going to have a amazing summer with plenty of wonderful experiences and new friendships. In this section, there will be two main topics, improvement and further development of our project, the PowerLeaf, and some of our project ideas generated in the startup phase to use for your project or start your creative thinking. Further Development of Our Project For those of you, that found interest in our project this year and would like to continue on improving it; this is the section you were looking for. We have listed the systems and the related information on theses needed for the device we envisioned, however you should not be limited to those. You are more than welcome to contact any of us regarding questions to the project, you can find our email addresses to each of us in the Team section of the Credits. Systems that didn’t work: Systems we didn’t work on, but should be implemented in the device: Ideas from Our Idea Generation List of ideas from our idea generation Credits Just like in movies, you get to meet the brilliant minds behind the project in the credits. Some might leave the cinema without reading the credits, but we hope you will continue to read ours, as it’s just as important and you will get to know us on a more personal level through this. We probably have more in common than you think. Behind every great team is a great amount of external attributions. The contributors have supported and inspired us, especially when things have been rough and deadlines near. Afterwards you can turn your attention to our collaborations, which was an amazing experience, this really shows of the true iGEM spirit.
Welcome to the team page, here you get to know us on a more personal level. As a team, we are 12 students from 8 different majors. As friends, we experienced the most amazing summer together, filled with various fun activities, both in- and outside the lab. To mention a few; we had road trips, dinners, Game of Thrones night and we even celebrated Christmas in July! We shared all of this with our amazing supervisors, for which we are truly greatful.
Emil Bøgh Hansen Study: Biology Emil Søndergaard Study: History Emil Vyff Jørgensen Study: Physics Ellen Gammelmark Study: Biochemistry and Molecular Biology Felix Boel Pedersen Study: Biochemistry and Molecular Biology Frederik Bartholdy Flensmark Neergaard Study: Biochemistry and Molecular Biology Frederik Mark 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 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 Project Synergism 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; "Alone we can do so little; together we can do so much" 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. Thanks to all the people that made this 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 decided to take advantage of our interdisciplinary team roster, and designed a wiki and ethics workshop to aid our fellow Danish teams.
Attending meetups Besides hosting our own meetup, we also attended several ones during our iGEM experience. The first of which, was the 5th Annual Biobrick Workshop in March, hosted by the Technical University of Denmark. This meetup not only gave us our first experience with Biobricks, but also worked as a foundation for friendships across the teams.
Further collaboration In 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.
‘Thank you for your time, we hope you enjoyed our wiki and project’. 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 of stories from our amazing iGEM summer.
lots of fun stories and pictures
All the best,PowerLeaf - a bacterial solar battery
Abstract
About Our Wiki
Since it’s a one page wiki, all of our text will be displayed on this page, which might seem overwhelming at first. Don’t worry though, to avoid unnecessary time consumption, the front page of the wiki will only contain the strictly necessary information. Whenever you need this additional information, you can always click a ‘read more’-link, which will display the information as a pop-up window. Once you have finished reading the in-depth details, you simply close the pop-up window, and continue right where you left off. We have prepared an example for you right here. Throughout the wiki, you might stumble upon this icon (book icon), which if you hover over it with the mouse, will show the references used for the associated statements.
I am fully aware of our readers being in a constant race against time, and I definitely identify with those who just want to read the essentials of the wiki as fast as possible. So, I went ahead and highlighted some of the important segments, making the entire wiki easy to quickly skim-read. Whenever you need my highlights, just go ahead and enable them in the top right corner. Go ahead and give it a try, it makes this wiki-nonsense so much easier to read. ;-)
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. 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’s one cat in the bag. You can find all attributions made to the project in the credits section of the wiki here. 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 in time.
Clearly state the Attributions – All attributions made to our project have been clearly credited in the credits section.
Improve and/or characterize an existing Biobrick Part or Device – Pending
Collaboration – We have collaborated with several teams throughout our project by taking part in discussions, meetups, answering questionnaires - we even hosted our first meetup for our fellow Danish teams. You will get to read all about all of 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 public engagement and education.
Improve a previous part or project – Pending
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 RelE (toxin), as this would make the bacteria unable to exit dormancy. To make them enter dormancy, it would require tight regulation of the RelB (anti-toxin). This information was used in the approach to light sensitive dormancy system.
Demonstrate your work – Requirement not fulfilled.
World Situation
Fortunately we learned through interaction with local experts that a great deal of people share our belief; that we ought to pursue the advancement 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.
We decided to pursue this goal by taking on the challenge to create a truly green solution, which will provide both an environmental-friendly source of energy, as well as a green aesthetic and naturalistic ambience to compliment a high quality of city-life.
Please keep scrolling if you wish to read more about our solution, or go straight to bioethics if you wish to read about why we not only could, but ought to do something about the current 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 creative thinking, public engagement, and collaborations. We worked with local city planners from our hometown Odense, in order to advance on this design and to provide other, changeable, designs.
Our vision was clear and ambitions were high, probably too high, considering the limited timeframe. So, at an early stage, we decided to focus on the following features:
Energy storing (E. Coli)
Project Design
Dormancy System
When not exposed to light, the photoreceptor PCB activates the phytochrome Cph1, thereby 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.
Different system-designs have been considered and tested thoroughly throughout the summer. This constitutes the foundation for how the design of the light induced dormancy system in E. coli has been optimized and the final approach shaped. Ultimately, the dormancy system, which is illustrated in figure #, was composed of the following parts:
Light Sensing System
The first construct containing the genes required for the light induced dormancy was designed as shown in figure #. (Reference to first figure) To confirm the functionality of the OmpR-regulated promoter, 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, but should be fusioned into the bacterial chromosome 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. is 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 is flanked by sequences that are homologous to part of the chromosome. The linear DNA fragment is 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 mediates the recombination. The fundamental concept of this approach is illustrated in figure #.
Originally, it was thought to place the antitoxin RelB under a constitutive promoter of appropriate strength. However, 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 when cloned into a low copy vector, and therefore this regulated promoter was chosen.
To simulate the RelB expression, a reporter system composed of the LacI-regulated, lambda pL hybrid promoter and GFP was assembled to identify the appropriate concentrations of IPTG required to induce the promoter on a low copy vector. During the cloning, it was discovered that the site between the LacI-regulated, lambda hybrid promoter and this particular reporter construct under the tested conditions had formed a hotspot for transposons. In the light of this finding, the ordinary LacI-regulated promoter, BBa_R0010, was chosen to express RelB. Experience from the previous projects shows that gene expression varies as a function of IPTG concentration only when the LacI-regulated promoter is expressed on a low copy plasmid. However, the promoter emerged unresponsive to regulation. Thus, 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 a all-or-none behavior when cloned into pSB1K3. Taking these result into consideration, the pBAD promoter was used in combination with the pSB3K3 vector for regulation the expression of the antitoxin RelB. Again, a reporter system was used to simulate the expression of RelB. The part, BBa_I6058, containing YFP controlled by pBAD was assessed on different vectors.
In the light of the system approaches reviewed in the preceding part, the final design, which is illustrated on figure # 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 on a low copy vector, and RelE controlled by the OmpR-regulated promoter on either a low copy vector or the chromosome.
[Bilag 1]
It was deduced, that when enhanced RelE production is implemented as a tool to make the bacteria dormant, the effect come easily. However, an additional implementation of RelB expression is found necessary to ensure that the bacteria enter an active state again.
The model showed that the system is sensitive to the RelE:RelB ratio as well as the total production of toxin. Implementation, with production rates in the vicinity of 50 and 35 molecules pr. min for RelB and RelE respectively, yields an acceptable effect: The bacteria lay dormant within the computed time, and re-enter an active state quickly. From the sensitivity to RelB production and RelE-RelB on activation time, it is evident that it will be challenging to implement an optimised system.
Carbon Fixation
Cellulose production and secretion
The ability for G. xylinus to produce, crystallise and secrete cellulose is controlled by genes in the cellulose synthase operon acsABCD. The acsABCD operon encodes four different proteins whereof AcsA and AcsB forms a dimer known as AcsAB. Together with AcsC, AcsAB is essential for biosynthesis of cellulose. AcsD has shown to be non-essential in production of cellulose, but absence of the protein results in decreased cellulose production, properly due to inappropriate crystallization of the polymer.
Other genera as E. coli excrete cellulose as a component of their biofilm. Even though cellulose production is intrinsic in E. coli, the in quantity the production is incomparable to cellulose production in G. xylinus. Indigenous E. coli is not capable of breaking down cellulose into a metabolisable energy source, but if the structural and water-holding polymer is enzymatically broken down into glucose residues, the cellulose polymer is a potent energy source. Read more here
The aim of this subpart of the project was to enhance cellulose production in the E. coli strain MG1655, to enhance the energy outcome of the entire system.
To enhance the cellulose production, parts containing the coding sequence of the Cellulose Synthase enzyme was transformed into E.coli strain MG1655. The original idea was to clone the entire cellulose synthase operon acsABCD under control of ptac. This was attempted many times, but with a total length over 9000bp the cloning was difficult, and unsuccessful. The plan was then to insert the cellulose synthase operon acsABCD into MG1655 on two plasmids, both controlled by the ptac promoter (http://parts.igem.org/Part:BBa_K864400). The catalytic domain, acsA, and the regulatory domain, acsB, both found in the part BBa_K1321334 (indsæt link), was attempted inserted into the vector psB1C3, carrying a chloramphenicol resistance cassette. Part K1321335 (indsæt link) containing acsC and acsD was inserted into the vector psB1A3, carrying an ampicillin resistance cassette. This was attempted many times, however, due to time constraints these clonings were also unsuccessful.
If the cloning of the cellulose synthase operon had been successful the cellulose production would have been tested using the fluorescent brightener 28... Read more here
Breakdown of Cellulose
Cellulose is a long polysaccharide consisting of β-1,4 linked D-glucose units. Many organisms, including E. coli, are incapable of producing enzymes able to deal with these strong β-linkages. To overcome this obstacle, the Cellumonas fimi developed to express two cellulases; the endo-β-1,4-glucanase (CenA) and the exo-β-1,4-glucanase (Cex) 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 break the amorphous structure of cellulose. This allows for the exoglucanase to break the β-1,4 linkages at every other D-glucose unit, thus releasing disaccharides 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.. Cellulose itself is practically too large to be transported across the cell membrane. This concludes that the breakdown of cellulose into cellobiose, must take place in the extracellular fluid.
The a-hemolysin transport system is an ABC transporter complex, consisting of the three proteins; 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.. This ABC transporter complex effectively transports intracellular hemolysin A (HlyA) to the extracellular fluid. This system can be utilised to secrete HlyA tagged proteins, using a peptide linker to fusion the protein of interest with HlyA. Once a protein is HlyA tagged, it can be recognized by the ATP-binding cassette HlyB, which will initiate transportation of the HlyA tagged protein to the extracellular fluid. 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 transported across the cell membrane, transportation of cellobiose is a common feature found in many organisms. An example is the E. coli, which 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.. Intracellular cellobiose can then be enzymatically broken down to glucose.
Through evolutionary events, many organisms have developed the ability to express enzymes capable of breaking the β-linkage of cellobiose. E. coli is known to express periplasmic β-glucosidase (BglX), which is known to have said feature by hydrolysing the cellobiose β-linkage Link til Bglx . The Saccharophagus degradans express an alternate enzyme, which can efficiently cleave the β-linkage in cellobiose. This is the so called cellobiose phosphorylase (Cep94A), which phosphorylates the cellobiose β-linkage resulting in its breakdown 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..
In the pursue to make E. coli utilize cellulose as its only carbon source, we based our project on the Edinburgh 2008 iGEM team project, who developed two Biobricks containing the CenA and Cex genes. We then utilised the a-hemolysin transport system by creating HlyA tagged CenA and Cex, using a linker peptide. Once tagged with HlyA, the cellulases can be secreted to the extracellular fluid, where they actively cleave cellulose to cellobiose units. To implement this system in E. coli, it would require the heterogeneous expression of HlyB, HlyD, CenA-HlyA and Cex-HlyA.
To accomplish this we ordered DNA synthesis of CenA and Cex, each tagged with HlyA using a linker peptide. The genes encoding HlyB and HlyD was retrieved from the BBa_K1166002 by phusion PCR. Using the aforementioned parts, we composed the following construct for the degradation of cellulose to cellobiose:
In the pursue to make E. coli utilize cellulose as its only carbon source, we based our project on the Edinburgh 2008 iGEM team project, who developed two Biobricks containing the CenA and Cex genes. We then utilised the a-hemolysin transport system by creating HlyA tagged CenA and Cex, using a linker peptide. Once tagged with HlyA, the cellulases can be secreted to the extracellular fluid, where they actively cleave cellulose to cellobiose units. To implement this system in E. coli, it would require the heterogeneous expression of HlyB, HlyD, CenA-HlyA and Cex-HlyA.
To accomplish this we ordered DNA synthesis of CenA and Cex, each tagged with HlyA using a linker peptide. The genes encoding HlyB and HlyD was retrieved from the BBa_K1166002 by phusion PCR. Using the aforementioned parts, we composed the following construct for the degradation of cellulose to cellobiose:
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, separated by a proton exchange membrane. Both the anode and the cathode in a MFC can use various forms of graphite as the base material. In the anode chamber of a MFC, 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, to generate needed ATP to maintain cellular life. This metabolic pathway also generates a release of electrons, carried by NAD+ in its reduced form NADH.
insert figure
Under aerobic conditions, the generated NADH will deliver its electron as part of the electron transfer chain, to return to its oxidised form NAD+. Under anaerobic conditions the electron transport chain will not be able to continue, which will cause the generated NADH to accumulate. As a consequence of accumulated NADH, the concentration of available NAD+ for glycolysis will decrease. This will drive the cell to do 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 a MFC will usually catalyse the reaction of 4 H+ + 2 O2 à H2O. The transfer of electrons from NADH to the anode can be executed in three different ways; 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.
insert figure
The redox shuttles use extracellular electron mediators which hold the advantage of not being limited by the surface area of the anode. However, it is restricted by the slow diffusion of the extracellular mediators. The direct contact electron transfer, in reverse to the redox shuttles, is strongly limited by the surface area of the anode, but the membrane bound cytochromes in direct contact with the anode, rapidly delivers the electrons. Bacterial nanowires are known to efficiently transfer electrons, much like 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 is. This is because bacterial nanowires are able to form complex networks of interacting nanowires in biofilm, to efficiently transfer electrons from distant microbes all the way to the anode using this network. 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.
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.. G. sulfurreducens is strictly anaerobic, as it would not be able to transfer its electrons to the environment in the presence of the highly reducing oxygen. Nanowires found in G. sulfurreducens is a type IV pilin polymer chain composed of PilA monomers, which can reach nearly 10 mm in length 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 compose a complex poorly understood system, which involves a long series of c-type cytochromes 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..
We then decided to work on optimisation of the G. sulfurreducens by increasing the electrical conductivity of its endogenous nanowires. To achieve this, we ordered synthesis of the PilA genes from G. metallireducens, which was used to create a Biobrick. From this Biobrick, a PCR product was made containing the chloramphenicol resistance cassette of the pSB1C3 backbone for later selection of recombinant G. sulfurreducens. The PCR product was ligated with PCR products retrieved from the 500 bp upstream and downstream regions of the chromosomal PilA genes of the G. sulfurreducens PCA strain. This was used to create the following linear DNA fragment, intended for homologe recombination into G. sulfurreducens:
Demonstration and Results
Parts
Notebook
SOPs and Protocols
Safety
Furthermore, our team participated in the 5th annual BioBrick workshop hosted by DTU BioBuilders. Here we engaged in a lab safety course before entering their lab. Both of these lab safety courses gave us the necessary knowledge to work safely with GMO, proper handling of waste and the according procedures in case of an emergency.
In the lab, we worked with several potentially harmful chemical agents such as DMSO (dimethyl sulfoxide), 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. We used a UV board to visualize bands in agarose gels. UV rays are carcinogenic when exposed frequently and for longer periods of time. 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 no longer than the necessary. GMOs were always handled wearing gloves, and all team members wore clean lab coats restricted to the laboratorial areas.
One of the biggest concerns would be the release of GMOs into nature. While the GMOs used aren’t pathogenetic, they would be able to share the plasmids containing antibiotic resistance selectors to other pathogenic bacteria. Antibiotic resistance in pathogenic bacteria, complicates the treatment of an infected individual, and could in tragic cases be the line between life and death. However small this scenario is, 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 natural balance, that we are aiming to restore with the development of the PowerLeaf.
To safely avoid these risks, there should be implemented several kill switch mechanisms into the final device. This could be performed by implementation of a light sensing system into the energy converting unit, which would turn on the kill switch if exposed to light. 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 actively function, could then have a kill switch which makes it completely dependant on presence of the container. This could be accomplished by having harmless molecules not naturally found in nature circulate in the system. Which should be required for the survival of the energy converting unit. A similar effect could be accomplished by making the energy converting and the energy 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.
Escherichia coli strains: K12, TOP10, MG1655, KG22, BW25113, ER25663127
Geobacter Sulfurreducens strain: PCA
Vectors
pSB1A2: An iGEM plasmid backbone carrying an ampicillin resistance gene
pSB1C3: An iGEM plasmid backbone carrying a chloramphenicol resistance gene
pSB1K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB3K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB4K5: An iGEM plasmid backbone carrying a kanamycin resistance gene
Bacteriophages
P1 phage, using its site-specific recombinase for transduction of E. Coli
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Bioethics
Integrated Practices
We will now walk you through our integrated human practices, so scroll on down to find out more about who we spoke with and how their input and advice came to influence our entire iGEM experience.
Furthermore the conversation with Mrs. Dienhart was also a source of inspiration in regards to our ethical and safety thoughts. The belief that while we ought to create a better more sustainable tomorrow for ourselves and future generations, we do not necessarily have to provide an exhaustive description of what that future should like, very much evolved from the changeability aspect, which was brought about by our conversation with Mrs. Dienhart.
Bolbro is an old neighbourhood in Odense historically known to be the home of the working-class, and 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 a reservation of approximately 1,6 million us dollars to renew its city-space and 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.
Mrs. Mortensen, as Mrs. Dienhart, also argued that a changeable design would be the optimal solution to fit the challenges, One faces in creating a vibrant, green city-ambience. As such a task depends on preferences, laws and needs. Instead a technology needs to be both flexible and accessible in order to successfully contribute to the process of creating a successful city environment. Furthermore we had a discussion with Mrs. Mortens about the creation of a prototype based on the wishes of Bolbro’s local citizens.
Having decided that the exterior of the device would be made entirely from plastic, we set out to.
Plastic is thought of as an undesirable material, due to the difficulties in its disposal. This is due to plastic being of a xenobiotic nature, making it generally recalcitrant to microbial degradation. This predicament is complicated further by biodegradable plastics being of a compensatory nature; sacrificing form-stability and strength for biodegradability. Following these concepts, we can identify the following set of criteria for our material:
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 in the 1970ies. A meeting was quickly arranged, where we fleshed out the 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 (kilde). The material, however, cannot fulfill the criteria on it’s own. Therefore, Mr. Christiansen suggested that we take a few liberties with it. In order to prevent the exposed part of the prototype from degradation by UV radiation, we will be adding certain additives to the surface of the exposed part. This doesn’t hinder the sunlight from entering the device and thus the bacteria, but just increases the UV-resistance of the material. During our consultations 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 GMOs inside would be exposed to the environment. The solution we came up with was the possible implementation of a kill-switch in the energy storage unit, making it vulnerable to light. Should the bacteria of said unit be exposed to sunlight, they would die, and since it’s counterpart in the solar cell unit would be dependent on the continued coexistence of the two units, the entire GMO system would be purged. With Mr. Christiansen’s help we designed the container for Cell 2 of the same material as Cell 1, albeit with an added compound. The container for Cell 2 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. Next, one must purchase the required material, which at above 1 ton would cost around 4-5.5 USD per kg. As such it’s an expensive material compared to others, but it’s longevity and durability means one would not be required to replace the devices for a long time. Lastly, we discussed the reusability of Polycarbonate, which Mr. Christiansen 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 SDU-Denmark teams as well as students from Odense. In extension of this, we hope that such a collaboration will help them see the benefits in collaborating with local agents.
Education & Public Engagement
Perspectives
As tools for genomic editing improves, the advancement of biological devices will conceivably become even more complex and independant. They will do so by introducing new metabolic pathways inspired from other organisms using genetic engineering. This could potentially allow the PowerLeaf to become completely independent through its self-replication, by producing their own essential nutrients directly from unwanted pollution in the environment. A process that would lead to cleaner cities, along with providing a natural solution to sustainable energy.
The PowerLeaf will be representing a natural leaf design, thus leading to a nature-in-city ambiance, which can have a soothing effect in the ever so stressful cities. Not only will the design represent a plant leaf, but some of the key functionality aspects of the device are inspired from those of a plant leaf. Hereby, we refer to photo synthesis and building cellulose as a biological product.
To Future iGEM Teams
Systems that did work:
Last but not least, don’t miss out on the ‘after-the-credits-clip’, which summarizes the fun we had during this wonderful iGEM experience. This is especially important, since you get the words ‘thank you for listening, we hope you enjoyed our wiki and project’ - we know you have been waiting impatiently to reach that part of the wiki.
Team
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 put my boots in the closet, in order to put on a proper lab coat doing iGEM. Besides my time in the lab I’ve also looked into how GMOs can influence the environment.
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: elgam15@student.sdu.dk
Why, hello there! My name is Ellen, and I spend most of my waking hours either in the lab with a pipette in my hand, or just outside it with a computer on my lap. You know.. Learn iGEM, live iGEM, love iGEM!
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 when the other miss me too much, I join them 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 make fun with the other teammates and tell bad dad jokes. Also I 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 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 such things most people like to do. During iGEM these interest has changed… I have been enslaved into the lab, and has realised 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 just yet.
E-mail: malta14@student.sdu.dk
Ey what up pimps, 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 hell 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, caused me to pull out most of my hair.
E-mail: sajo415@student.sdu.dk
Despite my favorite occupation being going into depth with theory, my main attribution to our project has primarily been running around in the lab. Luckily, there is a clear link between wet- and dry-lab. I am the smallest member of the SDU iGEM team, but I have definitely risen to the occasion.
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. When I’m not in the kitchen, busy making cakes for my teammates, you can find me in the lab, where I’m working on enhancing our systems cellulose production.
Attributions
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 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 bioethics, 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 past. To facilitate this exchange of knowledge on wiki development, we recruited our current supervisor Thøger Jensen Krogh, to hold presentations on how to design a good wiki. He was qualified for this task through his role as the designer of the SDU iGEM 2013 and 2014 team wikis, 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, which 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 a tradition. They suggested for all of us to meet again closer to the wiki deadline, to evaluate each team’s progress.
Our second meetup, the Nordic iGEM Conference, was hosted by the University of Copenhagen in June. The main focus of this meetup, was the traditional mini Jamboree. Participating in this gave us useful feedback from the judges, as well as from our fellow iGEM teams. This helped us greatly 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, learned from all the other great iGEM projects, and 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 from 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 scrutinized here.
We sought expertise from the Macquarie 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 the previous teams had experienced throughout their projects. During the skype conversation, we realised, that they could benefit from our knowledge on the electron transport pathways, that we used for our project.
We were also able to help the Stony Brook iGEM team by facilitating communication with members of the SDU iGEM team from 2016. Shortly after the European meetup, we received an email from the Cologne-Düsseldorf iGEM team regarding a postcard campaign, which we gave some feedback on.
During our project we received several questionnaires from fellow teams. We were delighted to help the teams by answering their questionnaires. The questionnaires were from:
Final Words
SDU-Denmark 2017
