editors highlights
ENERGY MADE BEAUTIFUL 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 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. A light-dependant system activates dormancy during nighttime to reduce energy lost by metabolism. The energy converting unit 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.
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 iGEMteam from the University of Southern Denmark. We have been waiting in great anticipation for the chance to tell you our story.
Bronze Medal Requirements 4/4 Register and attend – 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 2/4 Integrated Human Practices – Regarding the A Global Problem In the world of today, it is becoming increasingly important to ensure a sustainable future. 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; the intermittency and the diluteness problem. The intermittency problem describes the discontinuous energy production, along with inefficient storage. Whereas the diluteness problem is characterised as the resource demanding production of technical devices, such as solar cells and batteries. Meaning that a lack of resources eventually would eliminate 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 with an awareness 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 would come 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 with detailed stories. From this, the children would teach us a thing or two about the endless possibilities of GMO. See a selection of their amazing drawings here. 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-dependent 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”. 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.
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 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 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..
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.
Theory
Dormancy Optimises the Efficiency of the Bacterial Solar Battery 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 [2], 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 modelling, a hypothetical working system-design was devised.
For further information about our approach, read here. Approach Balancing Bacterial Dormancy Requires Accurate Regulation of the System
The Photocontrol Device was Placed under Control of a Constitutive Promoter
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.
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.
Modelling
Modelling of the RelE-RelB System is Essential to Avoid Irrevocable Dormancy 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. You can see the full results here.
Approach
The Gillespie algorithm is a way to calculate the evolution of stochastic functions; in this case cell concentrations. To use the algorithm, two things are required: For each time step two things are calculated, using the random number generator: The time before next reaction and which reaction occurs.
The time before next step is given by Approach
As an important aspect in the implementation of the RelE-RelB toxin-antitoxin system, modelling was performed. RelE is a toxin restricting growth by inducing a dormant state. This is inhibited by the antitoxin RelB, which forms complexes with RelE. Two different complexes are made: RelB2RelE and RelB2RelE2, containing 1 and 2 RelE molecules respectively [8], as seen in figure #.
For natural purposes, the half-life of RelB decreases significantly under starvation due to an Lon-protease [6], which shifts the equilibrium of RelB and RelE to a higher concentration of RelE. In a non-starvation situation, the interactions with the promoter keeps the amount of free RelE at a low concentration, thereby stabilising the system [1]. In our simulation, the shift in equilibrium is made by introducing additional translation of RelE.
Two different models were used with two different approaches. Firstly, it was seen how a given configuration of RelB and RelE production increased the RelE concentration and whether it could induce dormancy within 2 hours. Secondly, it was investigated how long it takes for each configuration to exit dormancy. That is, how long it takes for the free RelE levels to decrease again. Rates and Reactions The skeleton of the inherent system in the model was based on the article “Conditional cooperativity in toxin–antitoxin regulation prevents random toxin activation and promotes fast translational recovery” [1].
For the binding of the promoter, the operator is inhibited when bound with either RelB or 1-2 RelB2RelE, given that the operator has two binding sites [6]. The cell is assumed to have four chromosomes with one promoter on each, as this stabilises the inherent system considerably [1]. The systems exhibit similar behaviour, but with more noise, for fewer chromosomes.
Notice that all values are integers as the Gillespie algorithm works with discrete numbers of molecules. The values were chosen based on a stable equilibrium found by letting the model run a simulation of the inherent system over 450 minutes with different starting values.
Running the Model The model is using the Gillespie algorithm to give a stochastic view of the system, and it is run in MATLAB. The code uses an implementation by MATLAB user Nezar (https://se.mathworks.com/matlabcentral/fileexchange/34707-gillespie-stochastic-simulation-algorithm).
Theory
Carbon fixation in autotrophic organisms is responsible for the net fixation of 7×1016 g carbon annually [kilde 2]. 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 [kilde 6]. 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 [kilde 8]. 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 [kilde 8]. 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 [kilde 8]. The shell associated protein is essential for the biogenesis of the ɑ-carboxysome [kilde 12]. 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 [kilde 21]. For further information about the theory behind the carbon fixation, read here.
Theory
Carbon Fixation through the Calvin Cycle
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 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.
Approach
Engineering E. coli to Perform the Calvin Cycle
We succeeded in assembling both carboxysome parts in the composite part, BBa_K2449030, but for some inexplicable reason, this composite part likewise emerged difficult to clone with the Tac-promoter. Therefore, it was decided to focus at other aspects of the project and, as for the Calvin cycle part, keep this part theoretical henceforth.
Theory
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, which produces cellulose in large quantities of high quality [1]. 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 pentose phosphate pathway [2]. 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 [3].
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 [4]. 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 [5].
Approach
To link the two bacterial compartments of the PowerLeaf, an efficient way to store the harvested energy was required. Research led to the finding that storing the chemical energy in cellulose would be a suitable approach, since this is a polysaccharide that bacteria normally are unable to degrade [1]. 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 [2]. 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.
Approach
Optimised Cellulose Biosynthesis in E. coli
To enhance the cellulose biosynthesis, parts containing the coding sequence of the Cellulose Synthase enzyme were cloned into the E. coli strain MG1655. The original idea was to clone the entire cellulose synthase operon acsABCD into one vector under control of the Ptac, as seen in figure #. With a plasmid with a total length over 9000 bp, the cloning emerged difficult. After several unsuccessful attempts, a different approach was sought.
In this design, it was attempted to implement the acsABCD operon into E. coli MG1655 on two separate vectors, with both parts controlled by PtacBBa_K864400, ensuring equal expression levels of the parts. The AcsAB dimer, encoded in the part BBa_K1321334, was attempted to be inserted into the vector pSB1C3. The part BBa_K1321335, containing AcsC and AcsD, was inserted into the vector pSB1A3. Several combinations of the two parts and different vectors carrying different resistance cassettes were attempted, but unfortunately without success. Correspondence with a supervisor from the Imperial College London team, revealed that cloning with these parts had emerged difficult for them as well. Due to time constraints, it was decided to prioritise other aspects of the project and therefore keep this part theoretical henceforth.
Theory
Cellulose is a natural biopolymer used for a huge variety of biological purposes. 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 [kilde 4]. 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 converts cellulose to glucose in a two-step process, with cellobiose as the intermediate [kilde 7].
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
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 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 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 as shown in figure x; redox shuttles, direct contact electron transfer, and bacterial nanowires [kilde 7][kilde 8].
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 due to bacterial nanowires ability 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. [kilde 8]
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 [kilde 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 [kilde 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. 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 SOP01 - LA plates with antibiotic SOP02 - ONC E. Coli SOP03 - Gel purification SOP04 - Colony PCR with MyTaq SOP05 - Plasmid MiniPrep SOP06 - TSB transformation SOP07 - Fast digest SOP08 - M9 minimal medium SOP09 - Ligation SOP10 - Phusion PCR SOP11 - Bacterial freeze stock SOP12 - Making LB and LA media SOP13 - Agarose gel DNA SOP14 - Table Autoclave SOP15 - Preparing Primers SOP16 - PCR Protocol USER Cloning SOP17 - Excision and Ligation of PCR Product in USER Cloning SOP18 - Speedy Vac SOP19 - Preparing Eurofins sequencing samples SOP20 - Antibiotic stock production SOP21 - Electroporation SOP22 - P1 phage transduction SOP23 - Genome extraction SOP24 - Plasmid miniprep without kit SOP25 - NBAFYE Geobacter SOP26 - Electroporation Geobacter SOP27 - SDS-PAGE Protocol 1 - Biobrick assembly - Light Sensing System Protocol 2 - Biobrick analysis - Light Sensing System Protocol 3 - Biobrick assembly - RelE-RelB system Protocol 4 - Biobrick analysis - RelE-RelB Protocol 5 - Biobrick assembly - Dormancy System Protocol 6 - Biobrick assembly - Dormancy System Analysis Protocol 7 - BioBrick assembly - pOmpR characterisation Protocol 8 - Biobrick assembly - Carbon fixation Protocol 9 - Biobrick assembly - Cellulose biosynthesis Protocol 10 - Biobrick analysis - Growth experiment Protocol 11 - Biobrick assembly - Cellulose secretion Protocol 12 - Biobrick assembly - Cellulose consumption Protocol 13 - Biobrick assembly - Metallireducens PilA Protocol 14 - Cellulose degradation analysis - SDS-page Protocol 15 - Cellulose degradation - Cellulase screening 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.
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 dependehttps://2017.igem.org/Team:SDU-Denmark/testnt 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 the vision of the PowerLeaf, a vision we would love to see develop into reality. For this reason we have created an overview of the project, in the hope that it will benefit future iGEM teams. Additionally, it is aimed to assist prospective teams, 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, recyclable plastics and bacteria. This in turn, 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 technologies used in today’s synthetic biology, research groups are working on genetic code expansion. We had an interesting talk from postdoc Julius Fredens, about his work on genetic code expansion. Once a technology like this advances, 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, knowledge and new friendships. 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 are more than welcome to contact any of us regarding questions to the project. You can find each of our team members contact informations in the Team section in 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 closing credits. Some might leave the cinema without sitting through the credits, but we hope you will continue to sit through ours, as you will get to know us on a more personal level - we probably have even 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 were amazing experiences - after all 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.
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 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 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. We would like to thank 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 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 ones 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 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.
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 Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Vestergaard P, Rejnmark L, Mosekilde L. Osteoporosis is markedly underdiagnosed: a nationwide study from Denmark. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA. 2005;16(2):134-41. Thank you for your time, we hope you enjoyed our wiki and project!
lots of fun stories and pictures
PowerLeaf - a bacterial solar battery
Abstract
A Green Wiki
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 – Requirement not fulfilled.
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 of the light-dependent dormancy system.
Demonstrate your work – Requirement not fulfilled.
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 took on 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 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, 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)
Dormancy System
When not exposed to light, 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.
Light-Dependent Dormancy System
Photosynthetic bacteria draw energy from sunlight to drive the fixation of carbon, implying that the bacteria are not able to carry out carbon fixation in absence of light. However, the bacteria constantly metabolise and the carbon fixed during time of exposure to light, will be used as an energy source for the cells. To circumvent this, a photocontrol system created by the UT Austin iGEM 2004 team was used in combination with the RelE-RelB toxin-antitoxin system native to E. coli. In this way, a light-dependent dormancy system was implemented in E. coli.
The Photocontrol Device Mediates Light-Dependent Gene Expression
Plants and several photosynthetic microorganisms, such as cyanobacteria, contain signal transduction systems, which makes them capable of reacting to light [12]. This ability gives the organisms the opportunity to adapt and optimise the regulation of their metabolic rate in response to sunlight. This property is achieved by photoreceptors incorporated in their plasma membrane, of which phytochromes are the most abundant and well described [1].
Several two-component signal transduction systems evolved in E. coli enables it to respond to various external conditions, such as osmotic stress, lack of metabolites and other external stress factors. Nothing indicates that light initiates such a two-component signal transduction pathway in wild type E. coli [9]. The UT Austin iGEM 2004 team applied the light sensing property of phototrophs to an E. coli. By aligning different phytochromes with the intrinsic kinase EnvZ from E. coli they revealed a way to create a two-component system consisting of a photoreceptor with an intracellular indigenous regulator system found in E. coli. By establishing this system the bacteria acquired the ability to respond to red light [3]. The photoreceptor from phytochrome known as Cph1 was isolated from the cyanobacteria Synechocytis PCC6803. Cph1 has a fusion site, which can be used to combine it with the kinase EnvZ, from the EnvZ-OmpR kinase-regulator system, to form a two-domain receptor known as Cph8. The chromophore phycocyanobilin (PCB) absorbs light in the red region with maximal absorbance at 662 nm [c]. When heterogeneously expressed in E. coli, it can, in combination with the light receptor Cph8, be used to form a light-sensitive circuit, making E. coli able to respond to red light [2].
In situations where no red light is present, the photoreceptor PCB activates the phytochrome Cph1, thereby promoting kinase activity through the EnvZ kinase, illustrated in figure #. When the transcription factor OmpR is phosphorylated by EnvZ, expression of genes controlled by the OmpR-regulated promoter is initiated. Excitation of the PCB by red light, results in a situation, where EnvZ will not be able to phosphorylate the transcription factor OmpR. The lack of phosphorylated OmpR leads to no activation of the OmpR-regulated promoter, thereby preventing gene expression by this promoter.
RelE and RelB Comprise a Toxin-Antitoxin System in E. coli
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 [4]. 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 [5]. RelB neutralize the toxic effect of RelE through interaction between the two proteins. In situations of amino acid starvation, it is appropriate for the bacteria to halt the translation in order to avoid errors owing to absent amino acids. Consequently, one of the exciting factors for the expression of RelE is conditioned by amino acid starvation [5].
Whether the cell lie dormant in response to expression of RelE depends on the ratio of RelB and RelE present in the cell. Several studies have shown that RelB RelE form a complex with RelB:RelE stoichiometry of 2:1 [6,7], 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 [7]. To prevent free RelE circulating and discharging toxic effects in the cells under favorable conditions, studies in vivo have shown that RelB is present in 10x higher concentrations than RelE [6]. The heterologous induction of RelE could cause dissonance in the RelB:RelE ratio leading to serious consequences for the cells [a]. The bacteria are not killed when RelE is present in abundance, but high expression of the RelE gene makes awakening of the bacterial cells a challenge [b]. Hence, introducing a toxin to cells in a successful manner constitutes a challenge.
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 induced dormancy system in E. coli has been optimized and the final approach shaped. Ultimately, the light-dependent dormancy system, which is illustrated in figure #, 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 #.
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 #.
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 #. (Reference to first figure) 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 #, 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 [6]. 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 [7]. Using this technique, a short fragment of chromosomal DNA at the bacterial attachment site attB [8] 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 [7], which mediated the recombination. The fundamental concept of this approach is illustrated in figure #.
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 #, 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.
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, stochastic modelling 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. 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. You can read more about the model here .
Gillespie Algorithm
Δt=S-1log(r1-1)
Where S is the sum of the reaction rates and r1 is a random number between 0 and 1. This gives the time as if the system was one reaction with reaction rate S, using the random number to give a normal distribution.
The reaction is chosen proportionally to each individual reaction rate, using another random number. This way, reaction with high rates compared to other reactions will happen the most.
The reaction is then carried out. The new time is the previous time, plus the time of the reaction, and a new reaction can be calculated. This continues until the time reaches the wanted limit, or a specific number of reactions have occurred. It is necessary to have a limit on the number of reactions, as it is possible for the time steps to grow smaller and smaller. In this case the calculation time quickly become either immense or impossible.
Toxin/Antitoxin System
Both RelE and RelB are expressed from the same promoter, relBE. When only small amounts of RelE is present, RelB and RelB2RelE represses transcription through relBE, by binding to the operator.
At higher concentrations of RelE, the toxin mitigates this repression, by reacting with complexes bound to the operator sequence [1].
In E. coli with a size of 1-2 μm, each nM of concentration can be approximated to 1 molecule. Thus all units are converted to being measured in molecules, as this fits the premises of the Gillespie algorithm.
To simplify the model, the high affinity of RelE and RelB was used to ignore single RelB and only consider the dimers, RelB2[1][9]. Thus all mentions of relB in the model is for dimers.
RelB has a relatively low half-life at about 3-5 minutes [6], whilst RelE is stable and its half-life, here 43 min, is an effect from dilution caused by the bacterial growth [1]. During dormancy, growth is restricted and the RelE half-life is increased to 2000 min, which is approximately a day, as the dilution effect is no longer applicable.
The transcription rates of RelE and RelB are based on the concentration of RelE and RelB under stable conditions. Here, RelB is 10 times more prevalent than RelE [2]. Thus, RelB has been given a transcription rate 100 times higher than RelE, to make up for the higher half-life of RelE.
As the complexes are relatively stable, they were given the same half-life as. However, for RelE to become active in the inherent system under starvation, RelB in complexes must decay [1]. Therefore, the rate is set to a fourth of free RelB.
The initial values in the model were as below
Molecule
Initial number of copies
mrna
7
RelB
410
RelE
0
relB2relE
65
relB2rel2
11
Free operator sites
0
O(RelB)
2
O(RelB2RelE)
0
O(RelB2RelE)2
2
The implemented total production rates shown in the model might seem too high, as they range from 1-350 molecules pr. min, while the rates in the inherit system is effectively around 80-100 for RelB and 2-5 for RelE. The possibility of placing the system on high copy plasmids, however, makes the high total production values reasonable, as the individual RelE promoter will only need a very low production rate.
Considering the inherent toxin-antitoxin system activated under starvation, it is evident that the magnitude of RelE copies is around 50-80 molecules pr. cell. This makes it reasonable to believe, that the cell enter dormancy when a few tens of free RelE copies are present.
For a full list of the constants, read here [Bilag 2]
For the dormancy runs deterministic initial values were put in, and the system was run for 30 minutes without activating the inserted toxin promoter. This resulted in a stochastic distribution of initial values mimicking variations between cells. Analysis shows, that 30 minutes is enough for the model to find a stable distribution, which is realistic considering the growth cycle of an E.coli cell.
For activation runs, the data generated at the end of a sleep run was used as initial value and and deactivated the relE expression. When the concentration of free RelE decreases to below 15 copies, a cell is considered active. This value is probably set too low, but tests show marginal difference between 15 and 45 copies, where the lower limit is chosen to decrease uncertainty of the cells state.
All runs simulate 1000 cells, which should be sufficient to get stable averages. The model assumes well-mixed conditions in each cell, but considers each cell independently. Furthermore, the model has no cut-off for maximum values of RelE, as the exact relation between RelE concentration and hibernation state is unknown, yet a functional cutoff is found through activation times.
Carbon Fixation
Carbon Fixation
Carbon fixation in autotrophic organisms is responsible for the net fixation of 7×1016 g carbon annually, thereby being the most imperative biosynthetic process in nature [kilde 2]. Six different autotrophic pathways for carbon fixation have been discovered in a variety of organisms [kilde 1]. 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 [kilde 6]. The Calvin cycle, as this pathway is also called, can proceed under aerobic conditions, and only three enzymes and one microcompartment involved are heterologous to the gram-negative bacteria E. coli, making this the most obvious choice for the implementation of a carbon fixation pathway. In contrast, the 3-hydroxypropionate pathway for CO2 fixation would require the transfer of ten heterologous genes [kilde 7]. Furthermore, the reductive carboxylic acid cycle found in phylogenetically diverse autotrophic bacteria and archaea [kilde 4] and the noncyclic reductive acetyl-CoA or Wood-Ljungdahl pathway [kilde 5] require strict anaerobic conditions.
The Calvin cycle involves eleven enzymes, of which eight are intrinsic to E. coli. The three heterologous enzymes are RuBisCo, SBPase and PRK. The latter phosphorylates ribulose-5-phosphate to ribulose-1,5-bisphosphate. This is the substrate for RuBisCo, which catalyses the carboxylation, whereby glycerate-3-phosphate is produced. Later in the cycle, SBPase catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphate, which is later converted to ribulose-5-phosphate, completing the circle. The net effect of three full cycles is the conversion of three CO2 molecules into one molecule glyceraldehyde-3-phosphate, which can be used for energy production via glycolysis or polysaccharide biosynthesis. Separately, these enzymes have previously been heterogeneously expressed in E. coli using various donor species, such as wheat [kilde 16], the algae Chlamydomonas sp. [kilde 18 , kilde 17], and the cyanobacteria Synechococcus [kilde 15].
The Carboxysome Increases the Efficiency of the Carboxylation by RuBisCo
Many photosynthesising bacteria have developed CO2 concentrating mechanisms to increase the efficiency of the carbon fixation process. Cyanobacteria and many chemoautotrophic bacteria utilise organelle-like polyhedral bodies, that increase the internal concentrations of inorganic carbon 4000-fold compared to external levels [kilde 8]. These microcompartments, called carboxysomes, appear to have arisen twice during evolution and have undergone a process of convergent evolution [kilde 9]. The two types, designated ɑ and β, share main structural and functional features [kilde 9]. The ɑ-carboxysome consists of a proteinaceous outer shell composed of six different shell proteins designated CsoS1ABCD and CsoS4AB, and encloses RuBisCo, the shell associated protein CsoS2, and the enzyme carbonic anhydrase CsoS3. On average, ~250 RuBisCo molecules are localised within each carboxysome, and these are organised into three to four concentric layers [kilde 10]. The carbonic anhydrase converts HCO3-, which diffuses passively into the carboxysome, to CO2, thereby driving the continued diffusion of HCO3- into the microcompartment [kilde 8]. 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 prevail over oxygenation [kilde 8]. The shell associated protein is essential for the biogenesis of the ɑ-carboxysome [kilde 12]. The genes encoding the enzymes and shell proteins forming the ɑ-carboxysome from Halothiobacillus neapolitanus are clustered into the cso operon. This operon has been heterogeneously expressed in E. coli and its transcriptional regulation [kilde 19] and functionality [kilde 20] has been studied.
Energy and Electrons are Required for Carbon Fixation
For the Calvin cycle to proceed, energy in the form of ATP and electrons carried by NADPH are required. In photosynthesising organisms, such as plants and cyanobacteria, these constituents are provided by photophosphorylation performed by the photosystem complex. When engineering E. coli to perform photophosphorylation, 13 genes for the biosynthesis of chlorophyll a and 17 genes for the biosynthesis of photosystem II need to be heterogeneously expressed. Several attempts have been made at expressing part of it, such as the psbA gene [kilde 13] and the 18-kDa protein of photosystem II [kilde 14], both of which was successful.
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 catalyse light-activated proton efflux across the cell membrane and thereby drive ATP synthesis. In contrast to photosystems, the process involving proteorhodopsin is anoxygenic and generates no NADPH vital for the Calvin cycle to proceed [kilde 21].The heterologous expression of this light-powered proton pump in E. coli enabled photophosphorylation when the bacteria were exposed to light [kilde 22], and even generated a proton motive force, which turned the flagellar motor, yielding light-dependent motility [kilde 21].
Carbon Fixation
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. In correspondence with the 2014 Bielefeld iGEM team, who worked with a similar subpart of their project, we discussed the cloning of these genes and received two crucial parts. The first of this was BBa_K1465214, containing RubisCO from Halothiobacillus neapolitanus and PRK from Synechococcus elongatus under the control of a composite promoter controllable by IPTG. The second was BBa_K1465228, which contained SBPase from Bacillus methanolicus. Through the concurrent expression of these parts in E. coli, all enzymes required to fixate CO2 through the Calvin cycle were present in the cells.
The first approach was to assemble both parts on one plasmid by molecular cloning, as shown on figure #. In doing this, it was discovered that the BBa_K1465214 part was missing roughly 1 kbp when digested with standard restriction enzymes with recognition sites within the BioBrick prefix and suffix. Sequencing of the part revealed that the part was missing the entire promoter sequence of 1239 bp. Consequently, the assembly of the parts additionally required the cloning of a promoter. For this purpose, the Tac-promoter (Ptac) from the part BBa_K864400 was chosen, as this is commonly used to overexpress genes in E. coli. With the objective to place the two Calvin cycle parts on different vectors, it was attempted to clone Ptac in front of both parts. However, this did not succeed and it was decided to prioritise other aspects of the project and therefore keep this part theoretical henceforth.
Implementing the Carboxysome in E. coli to Increase Carbon Fixation Efficiency
The implementation of the microcompartment carboxysome in the test organism can increase the efficiency of the carbon fixation process substantially. As for the Calvin cycle parts, we corresponded with the 2014 Bielefeld iGEM team on their experience of the implementation of the carboxysome, and received the two parts that together contained the cso operon from Halothiobacillus neapolitanus. These parts were BBa_K1465204, containing csoS2, and BBa_K1465209, containing csoS3, csoS14, and csoS1D. As part of the optimisation, we aimed to combine these parts into one part containing the entire cso operon, as shown on figure #. Furthermore, a promoter was required to drive the gene expression. For this purpose, Ptac was chosen, as for the genes encoding the Calvin cycle enzymes.
A fitting approach to verify the expression of the genes involved in carbon fixation, could consist of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and quantitative PCR (qPCR). For this, one could build upon the experience obtained by the 2014 Bielefeld iGEM team.
In order to test the functionality of the parts we had hoped to assemble, we had reached out to Nordcee, which is a major research group within the Department of Biology at SDU. Our hope was to compare the carbon fixation rates for cultures expressing the Calvin cycle enzymes alone and in combination with the carboxysome, whereby the effectivity of the microcompartment also would have been assessed. In cooperation with Nordcee, measurements of CO2 decrease in an airtight culture would have been carried out with an infrared CO2analyser. Another test approach would have been using 14CO2, and then measure the amount our organism had taken in.
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 secretes cellulose and forms an interconnected cellulose pellicle around the cells [6], as illustrated in figure #.
Cellulose Biosynthesis
If the cloning of the acsABCD operon had been successful, the cellulose biosynthesis would have been tested using fluorescent brightener 28. This is a colourless organic compound that fluoresces with a bright blue color under ultraviolet radiation, and it is used as a fluorescent brightening agent for polyamide and cellulose fabrics. Fluorescent brightener 28 binds non-specifically to polysaccharides with β-1,3 and β-1,4 linkages, of which the latter is present in cellulose [3].
Breakdown of Cellulose
Cellulose is a long polysaccharide consisting of β-1,4 linked D-glucose units. Many organisms, including E. coli, lack the enzymes able to degrade these strong β-linkages. To overcome this, the C. fimi developed two cellulases, namely the endo-β-1,4-glucanase and exo-β-1,4-glucanase, respectively encoded by the cenA and cex genes [kilde 7]. 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 [kilde 8], as illustrated in figure #. Cellulose itself is too large to be transported across the bacterial cell membrane. 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) [kilde 3]. The ABC transporter complex effectively transports 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 recognized by the ATP-binding cassette HlyB, which will initiate transportation of the HlyA-tagged protein to the extracellular fluid, as seen in figure # [kilde 3, kilde 6].
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, which utilises the membrane protein lactose permease (LacY) [kilde 5]. In the cytosol, cellobiose is enzymatically catabolised.
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, by hydrolysing the cellobiose β-linkage[kilde 1]. Saccharophagus degradans expresses a different enzyme, which 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 [kilde 5], as seen in figure #.
In the endeavour to engineer E. coli to utilise cellulose as it’s 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-glucanase, 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 on figure #.
The Edinburgh 2011 iGEM team team created a BioBrick encoding periplasmic β-glucosidase endogenous to E. coli, proposed to increase its efficiency at degrading cellobiose to glucose. However, it seems that the enzymatic activity of bglX has faded as a result of evolution, rendering E. coli incapable of surviving solely on cellobiose. So even though E. coli can absorp cellobiose, it is not able to survive with this as its only carbon source.
To solve this issue, we decided to synthesise a cep94A Biobrick, intended to make E. coli capable of effectively surviving on cellobiose. To achieve this we composed the following construct:
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 as illustrated in figure y. 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 [kilde 8]. 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.
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 [kilde 3]. 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 [kilde 4]. The proteins required for the effective transfer of electrons by nanowires is a complex and poorly understood system, which involves a long series of c-type cytochromes [kilde 6].
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 homologous recombination into G. sulfurreducens:
Demonstration and Results
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!
Week 45
Wiki freeze!!!
SOPs and Protocols
SOPs
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.
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
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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. An ability that would occur when the bacteria are engineered to produce their own essential nutrients directly from unwanted pollution in the environment. A trait that would lead to cleaner cities, along with providing a natural solution to sustainable energy.
To Future iGEM Teams
Systems that did work:
Finally, we do not want you to miss out on the ‘after-the-credits-clip’, which summarises the fun we had during this wonderful iGEM adventure. This is especially important, as this is 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 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: 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: 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.
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. 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.
Our second meetup, the Nordic iGEM Conference, was hosted by the University of Copenhagen, UCopenhagen, 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 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 **link to report*. Due to our team being the most green team TU-Dresden asked us to run the iGEM Goes Green project in 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 the 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. The questionnaires were from:
Attributions
Articles
Websites
Books
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 of stories from our amazing iGEM adventure.