Revision as of 20:11, 22 October 2017

PowerLeaf - a bacterial solar battery

ENERGY STORED IN CELLULOSE • LIGHT SENSING DORMANCY SYSTEM • OPTIMISED NANOWIRES

Abstract

The PowerLeaf introduces a novel solution for long-term storage of solar energy, thus becoming an alternative to solar cells. This is accomplished without the use of environmentally harmful resources. The device is designed to resemble a plant leaf aimed to provide a nature-in-city ambience. This hypothetical implementation of the PowerLeaf in an urban environment was developed through public engagement and collaboration. The bacterial solar battery is composed of an energy storing unit (a) and an energy converting unit (b). The energy storing unit (a) is defined by a genetically engineered Escherichia coli that fixates carbon dioxide into the chemically stable polymer cellulose. A light sensing system activates dormancy during nighttime to reduce energy lost by metabolism. The energy converting unit (b) uses genetically engineered Escherichia coli to consume the stored cellulose. Retrieved electrons are transferred by optimised nanowires to an anode resulting in an electrical current.

About Our Wiki

Functionality on the Wiki

Hi there, and welcome to our iGEM experience. I’m the creator of this wiki, and this year I decided to make it a one-page wiki - to make it stand out from the crowd. All you have to do is keep scrolling, and you will be guided through the project. You will always be able to follow your process through the wiki, by looking at the navigation bar. Whenever you want, you can click on one of the tabs, to get an overview of the sections associated with it. If you know where you want to go, you can click the section-links in each of the tab menus, and they will get you there immediately.
Since it’s a one page wiki, all of our text will be displayed on this page, which might seem overwhelming at first. Don’t worry though, to avoid unnecessary time consumption, the front page of the wiki will only contain the strictly necessary information. Whenever you need this additional information, you can always click a ‘read more’-link, which will display the information as a pop-up window. Once you have finished reading the in-depth details, you simply close the pop-up window, and continue right where you left off. We have prepared an example for you right here. Throughout the wiki, you might stumble upon this icon (book icon), which if you hover over it with the mouse, will show the references used for the associated statements.
I am fully aware of our readers being in a constant race against time, and I definitely identify with those who just want to read the essentials of the wiki as fast as possible. So, I went ahead and highlighted some of the important segments, making the entire wiki easy to quickly skim-read. Whenever you need my highlights, just go ahead and enable them in the top right corner. Go ahead and give it a try, it makes this wiki-nonsense so much easier to read. ;-)

A Green Wiki

Our project is all about ensuring a greener and more sustainable future for ourselves and the coming generations. This of course meant, that our wiki had to to follow this pursue. CO₂NeutralWebsite sponsored our wiki with a CO₂ quota equal to the amount of CO₂ produced, by having the wiki running until 31-Oct-2018. This does not mean the wiki is CO₂ neutral, but that the quote equal to its pollution is bought. Buying a quota means, that other companies won’t be able to buy this CO₂ quota, thus, forcing them to improve their environmental policies if they wish to become CO₂ neutral.

Introduction

Welcome to our wiki! We are the IGEM team from the University of Southern Denmark. We have been waiting with great anticipation for the chance to tell you our story.
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

Bronze Medal Requirements 4/4

Register and attend – Our team applied 2017-03-30 and got accepted 2017-05-04. We had an amazing summer and are looking forward to attend the Giant Jamboree!
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

Silver Medal Requirements 3/3

Validated part/contribution – 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.

Gold Medal Requirements 3/4

Integrated Human Practices – Regarding the development and implementation of the device, we reached out to and remained in contact with city planners from our hometown throughout our project. This regarded advice and conversations on anything from the possible design, value, safety, use, placement and plastic type of our device. We also made sure to integrate the findings of said conversations into our overall project. Additionally we focused on demonstrating this process on our wiki; in order to inspire future iGEM teams.
Improve a previous part or project – Pending
Model your project – Through extensive modelling we have learned that it is possible to regulate bacterial dormancy. However, the modelling showed that it would be inadequate to only regulate RelE (toxin), as this would make the bacteria unable to exit dormancy. To make them enter dormancy, it would require tight regulation of the RelB (anti-toxin). This information was used in the approach to light sensitive dormancy system.
Demonstrate your work – Requirement not fulfilled.

World Situation

A Global Problem

redo the approach in this section

In a Local Environment

We are a team of young adults raised to be aware of climate changes and the potential limitations to the continuation of our way of life. We are a generation that appreciates open source and shared information. A generation that has been encouraged to constantly challenge the ideas of yesterday. With this in mind, we decided the best solution to the eventual energy crisis would be to seek out experts, the general public, even children, in order to rethink the current notion; that the only way to save our planet is to compromise our living standards.
Fortunately we learned through interaction with local experts that a great deal of people share our belief; that we ought to pursue the advancement of low energy cities with a high quality of life. In fact, we even discovered that our own hometown Odense wants to be the greenest, most renewable city in Denmark by 2050.Odense municipality’s website, regarding their politics on the current climate changes.
We decided to pursue this goal by taking on the challenge to create a truly green solution, which will provide both an environmental-friendly source of energy, as well as a green aesthetic and naturalistic ambience to compliment a high quality of city-life.
Please keep scrolling if you wish to read more about our solution, or go straight to bioethics if you wish to read about why we not only could, but ought to do something about the current energy crisis.

Inspiration

Our early ideas were reviewed after attending the Danish Science Festival, where we met several young minds with creative and inspiring ideas. The children would come to our booth with their parents to learn about bacteria, GMO, ethics and iGEM. After which, they would attend our “Draw-a-Bacteria”-competition. While drawing their own unique bacteria, they would present us with detailed stories about their design.

See a selection of their amazing drawings here.

Through this, we felt inspired and decided to revise our ideas. They even inspired the physical design for our original prototype; the PowerLeaf.

Our Solution

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.
The device was originally designed to resemble a plant leaf aimed to provide a nature-in-city ambience. This hypothetical implementation of the PowerLeaf in an urban environment was developed through creative thinking, public engagement, and collaborations. We worked with local city planners from our hometown Odense, in order to advance on this design and to provide other, changeable, designs.
Our vision was clear and ambitions were high, probably too high, considering the limited timeframe. So, at an early stage, we decided to focus on the following features:

Light-dependant dormancy system
Converting CO₂ to glucose
Biosynthesis and secretion of cellulose produced from glucose
Converting cellulose to glucose
Extracellular electron transfer

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:
Energy storing (E. Coli)

Light-dependant dormancy system
CO₂ fixation
Cellulose biosynthesis and secretion

Energy converting (G. Sulfurreducens)

Breakdown of cellulose
Extracellular electron transfer

Once you reach each of the 5 systems in the 'Project Design'-section, you will first be given a short introduction to the underlying theory, which you will be able to expand on, by pressing “read more”. Which in turn will open a pop-up window with the additional information. After the theory, you will be given the approach used in each of the respective systems for the project. Before continuing on to the next system. To make things easier on you, we have developed icons to each of the above systems which will be used throughout the rest of the wiki.

Project Design

overview picture, fix later, in a hurry, soft wiki

Light Sensing System

Theory

Cyanobacteria contain signal transduction systems, thereby making them capable of sensing and respond 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):12834-12839. doi:10.1073/pnas.1303371110.. 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 http://www.sciencedirect.com/science/article/pii/S1084952100902064?via%3Dihub. In 2004 the Austin iGEM team made a light response system consisting of a photoreceptor combined with an intracellular indigenous regulator system https://www.ncbi.nlm.nih.gov/pubmed/16306980. EnvZ and OmpR makes up the two-component system naturally found in E. coli. The photoreceptor from phytochrome known as Cph1 was isolated from the cyanobacteria Synechocytis PCC6803. Cph1 has functional combination sites, which combined with the kinase EnvZ forms a two-domain receptor, known as Cph8. Activation of Cph8 is mediated by the phycocyanobilin, PCB that is sensitive to red light.
When not exposed to light the photoreceptor PCB activates the phytochrome Cph1, thereby promoting kinase activity through the EnvZ kinase. When the 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.

Using the photocontrol device to set up a 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 https://www.ncbi.nlm.nih.gov/pubmed/9767574. 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. The indigenous cause of RelE expression is amino acid starvation https://www.ncbi.nlm.nih.gov/pubmed/12526800. 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:RelE forms a complex with RelB:RelE stoichiometry of 2:1 https://www.ncbi.nlm.nih.gov/pubmed/19747491https://www.ncbi.nlm.nih.gov/pubmed/18532983. 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 https://www.ncbi.nlm.nih.gov/pubmed/18532983. Read more here

Theory

To introduce an efficient energy harvesting system, the E. coli should lie dormant during nighttime, thereby reducing the energy used for the bacteria’s basic metabolism. For this purpose a photocontrol system created by a former iGEM team is used in combination with a toxin-antitoxin system found in E. coli, to make the bacteria able to respond to the environmental exposure of light.
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 photoreceptors. [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 Austin iGEM team from 2004 applied the light sensing property of phototrophs to an E. coli [10][11]. 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 phycocyanobilin, PCB, which is sensitive to red light and only expressed in genetically engineered E. coli, 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 of no exposure to light, the photoreceptor PCB activates the phytochrome Cph1, thereby promoting kinase activity through the EnvZ kinase. Illustrated in figure #. When the the transcription factor OmpR is phosphorylated by EnvZ, expression of genes regulated by the OmpR-regulated promoter is initiated. Excitation of the PCB by red light, results in a situation, where EnvZ won’t 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.
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 acids 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 forms 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]

Approach

In 2004 the Austen and UCSF iGEM teams created a device sensitive to light in the Coliroid project. Since the creation of this light sensing system, many iGEM teams have successfully implemented the device in their projects. In this project, this system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. As the RelE-RelB system requires tight regulation [2], different systems have been duly considered in the endeavours to find a way to induce RelE without completely paralysing the cells. Several different promoters have been considered in combination with different vectors. By modelling the toxin-antitoxin system and testing different approaches, a suitable and hypothetical working system design has been implemented.
Different system designs have been considered and tested thoroughly throughout the summer. This constitutes the foundation for how the design of the light induced dormancy system in E. coli has been optimized and the final approach shaped. The finite approach of the system in ended up consisting the following elements. The design appears from the figure below.

The toxin gene RelE controlled by the OmpR-regulated promoter with the pSB1C3 antibiotic resistance cassette is recombined onto the bacterial chromosome.
The photocontrol device under control of the PenI-regulated promoter cloned onto the high copy pSB1A2 vector.
The antitoxin RelB under control of the AraC promoter cloned onto the low to medium copy pSB3K3 vector.

Approach

The genes needed for inducing dormancy when the bacteria are not exposed to light are found in the photocontrol device part BBa_K519030. This part is composed of three genes, named ho1, pcyA, and cph8, which all are essential for the cells ability to respond to light. The photoreceptor Cph1 contains a chromophore, called phycocyanobilin, which absorbs light in the red region with maximal absorbance at 662 nm [3]. When the cells are not exposed to red light, activation of Cph1 takes place. Active Chp1 causes the EnvZ-OmpR complex to autophosphorylate and activate an OmpR-regulated promoter sequence. In the first envisaged design of the light-dependent dormancy system, the aim was to clone the photocontrol device K519030 under control of a constitutive promoter, RelE under control of the OmpR-regulated promoter and RelB under control of a constitutive promoter into high copy BioBrick assembly plasmid pSB1C3, as seen below:

From the constitutive promoter family [4] 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 RFP expression correlated with the strength of the promoter.

The data obtained from fluorescence microscopy revealed that the weak promoter had a high level of noise, causing a non-uniform expression throughout the cells [5]. Gene expression was more uniform in the bacterial cells containing the medium promoter BBa_J23110. The other medium strength promoter BBa_J23106, likewise had a phenotypical uniform level of gene expression, but less consistent than observed for BBa_J23110. Gene expression in cells with BBa_J23102 displayed strong gene expression and a low level of noise was observed. (Pictures obtained from fluorescence microscopy)
The photocontrol device genes was, on the basis of the above mentioned promoter test, placed under control of the strong promoter BBa_J23102. During the testing of the constitutive promoters the project made by William and Mary iGEM 2015 was studied with strong interest. After reading the results obtained by their team, it became clear that placing the antitoxin RelB under control of an controllable promoter would be a more appropriate choice in this system, instead of using one of the constitutive promoters. The William and Mary iGEM 2015 team found that the LacI-regulated, Lambda pL hybrid promoter, BBa_R0011 had a low level of noise and therefore it was chosen to regulate the expression of RelB.
The first construct containing the genes required for the light induced dormancy was designed as shown in figure #. (Figure of the first part construction, the one without anything in the genome #1) A midway test on the plasmid containing the photocontrol device followed by the OmpR-regulated promoter controlling expression of a reporter gene indicated a dysregulation of the OmpR-regulated promoter. Thorough research on the light regulated system lead to the finding that the OmpR dependent promoter should be fusioned into the bacterial chromosome, to obtain a regulated inducement of the gene under control of the OmpR dependent promoter [6]. Whereas each bacteria only contains one chromosome, the standard iGEM plasmid pSB1C3 has a copy number of 100-300 per cell. The statement that the OmpR-regulated promoter was leaky when cloned into a high copy vector, would explain the obtained results, as it is not regulated properly in this situation. A neat solution to this issue could be to fuse the OmpR-dependent promoter with the RelE gene onto the chromosome of the test strain. The design of the genes cloned into the plasmid was changed so that the photocontrol genes under control of the strong constitutive promoter was cloned into the same vector as RelB controlled by the LacI-regulated, Lambda pL hybrid promoter.

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 is replaced with a linear DNA sequence encoding the OmpR dependent promoter, the controlled gene, and an antibiotic resistance cassette. The linear DNA sequence is flanked by fragments that are homologous to part of the chromosome. The linear DNA fragment is electroporated into bacteria containing the red λ recombinase encoding plasmid [7], allowing the recombinase to mediate the recombination.

Inducing dormancy and bringing the bacteria back to a metabolic active stage is like balancing on a tightrope, as mentioned in the theory. To account for this, a system was created to simulate the gene expression of the RelE:RelB system under control of the OmpR- and LacI-regulated promoters. Furthermore, mathematically modeling was performed to prognosticate the system and simulate the interactions between the toxin and antitoxin (Link to the model page). As reporter genes, fluorescent proteins were chosen, as these do not have any significant influence on the viability of the cells. Red fluorescent protein, RFP, was placed under control of the OmpR-regulated promoter analogous to RelE. Green fluorescent protein, GFP, equivalent to RelB in the dormancy system intended to be controlled by the LacI-regulated, Lambda pL hybrid promoter.
During the cloning of GFP under control of the Lambda pL hybrid promoter, it was noticed that the part yielded three bands when digested with EcoRI and PstI, one at 2000 bp corresponding to the backbone, one band at 950 bp which corresponds with the proper length for the part, and a third band occurred with a length just above 1000 bp. (Paste a picture of the gel). The BioBrick was sequenced to identify the sequence giving cause for the band with the unaccountable length. The sequencing result revealed a repeated sequence inserted between the LacI-regulated, Lambda pL hybrid promoter and the GFP gene, encoding a transposable element. The part was tried assembled all over, with a similar result. This indicates that the site between the LacI-regulated, Lambda hybrid promoter abd GFP under the tested conditions form a hotspot region for transposons. In the light of this finding the ordinary LacI promoter was selected to express RelB and GFP in the prognosticate color system. Experiences in usage of BBa_R0010 revealed that when the promoter is cloned into a high copy plasmid the promoter is not repressible. The test results from the previous years indicate that gene expression varies as a function of IPTG concentration only when the LacI promoter is expressed on a low copy plasmid.

The LacI promoter controlling GFP and RelB was on basis of these experiences cloned into the low copy BioBrick standard vector pSB4K5. The genetic design of the system at this state appears from the figure above. Despite of the experience with gene expression controlled by the inducible LacI promoter in a low copy vector, the transformed into KG22, an E. coli strain with overexpressed LacI, all emerged phenotypical green both induced and uninduced with IPTG. This observation indicated that the BioBrick was not operating as intended. Rethinking the design of the BioBricks responsible for the light-dependent dormany lead to the use of another inducible promoter. The HKUST-Rice iGEM 2015 showed that the AraC promoter in combination with the the low to medium copy plasmid pSB3K3 showed a gradually increase in gene expression, whereas their results indicates that the AraC promoter cloned into pSB1K3 exhibit all-or-none behavior. Taking these result into considerations the AraC promoter was used in combination with the pSB3K3 vector for regulation the expression of the antitoxin RelB.
For so far inexplicable reasons the photocontrol device emerged difficult to clone with the strong constitutive promoter. Both the digestion, the ligation, and the transformation process was tried optimized, with very few successful clonings, and all the times the correct assembly was obtained, the BioBrick was not reproducibly purifiable. Getting around 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. Both were tested using composite parts of the promoters with yellow fluorescent protein (YFP), I6102 and I6103 respectively, and fluorescence microscopy. The obtained results showed that the PenI promoter facilitated very strong expression of the controlled genes, whereas the expression by Mnt-regulated promoter was considerably lower. On basis of these findings the photocontrol genes were placed under control of the PenI-regulated promoter instead of the strong constitutive promoter from the constitutive promoter family.

CO₂ fixation

Theory

Carbon fixation in autotrophic organisms is responsible for the net fixation of 7 × 1016 g carbon annually (Berg 2011). 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 (Bowien et al. 1987). Out of the eleven enzymes needed for the Calvin cycle, only three are heterologous to E. coli, namely; ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase (PRK). By the concurrent heterologous expression of the three genes encoding these enzymes, E. coli can be engineered to perform the full Calvin cycle.

The carboxysome is a microcompartment utilised by many chemoautotrophic bacteria, including cyanobacteria, as a CO₂ accumulating mechanism to increase carbon fixation efficiency. This organelle-like polyhedral body is able to increase the internal concentrations of inorganic carbon by 4000-fold compared to the external concentration (Mangan and Brenner 2014). 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 enclose RuBisCo, the shell associated protein (CsoS2) and the enzyme carbonic anhydrase (CsoS3). In the proteobacteria Halothiobacillus neapolitanus, these genes are clustered into the cso operon. The carbonic anhydrase converts HCO3-, which diffuses passively into the carboxysome, to CO2, thereby driving the continued diffusion of HCO3- into the microcompartment (Mangan and Brenner 2014). The increased CO2 concentration in the vicinity of RuBisCo increases the rate of carbon fixation by saturating the RuBisCo enzyme and increasing the CO2 to O2 ratio, enabling carboxylation to dominate over oxygenation (Mangan and Brenner 2014). The shell associated protein is essential for the biogenesis of the ɑ-carboxysome (Cai et al. 2015).

For the Calvin cycle to proceed, energy in the form of ATP and electrons carried by NADPH are required. The photosystems are complexes in photosynthesising organisms that can supply this by photophosphorylation. To engineer E. coli to do photosynthesis, 13 genes is needed for the assembly of chlorophyll a and 17 genes for the assembly of photosystem II, which needs to be heterogeneously expressed. An alternative process, in which a diverse array of phototrophic bacteria and archaea harvest energy from light, is through a retinal-containing protein called proteorhodopsin, which catalyses the light-activated proton efflux across the cell membrane and thereby drive ATP synthesis. Opposed to the photosystems, the proteorhodopsin is anoxygenic and generates no NADPH, which is crucial for the Calvin cycle to proceed (Walter et al. 2007). Read more here

Theory

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 (Berg 2011). Six different autotrophic pathways for carbon fixation have been discovered in a variety of organisms (Ducat and Silver 2012, Berg 2011). 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 (Bowien et al. 1987). The reductive pentose phosphate cycle, as the Calvin cycle 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 (Mattozzi et al. 2013), the reductive carboxylic acid cycle found in phylogenetically diverse autotrophic bacteria and archaea (Hügler et al. 2005) and the noncyclic reductive acetyl-CoA or Wood-Ljungdahl pathway (Ragsdale and Pierce 2008), require strict anaerobic conditions. The Calvin cycle involves eleven enzymes, of which eight are intrinsic to E. coli. The three heterologous enzymes are ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), sedoheptulose-1,7-bisphosphatase (SBPase) and phosphoribulokinase (PRK), as seen on figure #. 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 carbon dioxide 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 (Dunford et al. 1998), the algae Chlamydomonas sp. (Tamoi et al. 2005, Vira et al. 2016), and the cyanobacteria Synechococcus (Parikh et al. 2006).

Carboxysome

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 (Mangan and Brenner 2014). These microcompartments, called carboxysomes, appear to have arisen twice during evolution and have undergone a process of convergent evolution (Rae et al. 2013). The two types, designated ɑ and β, share main structural and functional features (Rae et al. 2013). The ɑ-carboxysome consist of a proteinaceous outer shell composed of six different shell proteins designated CsoS1ABCD and CsoS4AB, and encloses RuBisCo as well as the shell associated protein (CsoS2), and the enzyme carbonic anhydrase (CsoS3), as seen on figure #. On average, ~250 RuBisCo molecules are localised within each carboxysome, and these are organised into three to four concentric layers (Iancu et al. 2007). The carbonic anhydrase converts HCO3-, which diffuses passively into the carboxysome, to CO2, thereby driving the continued diffusion of HCO3- into the microcompartment (Mangan and Brenner 2014). The increased CO2 concentration in the vicinity of RuBisCo increases the rate of carbon fixation by saturating the RuBisCo enzyme and increasing the CO2 to O2 ratio, enabling carboxylation to dominate over oxygenation (Mangan and Brenner 2014). The shell associated protein is essential for the biogenesis of the ɑ-carboxysome (Cai et al. 2015). 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 (Cai et al. 2008) and functionality (Bonacci et al. 2012) has been studied.

Energy an electron source

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. To engineer 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 (Efimov et al. 1994) and the 18-kDa protein of photosystem II (Kuwabara et al. 1995), both of which had success.
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 (Walter et al. 2007). The heterologous expression of this light-powered proton pump in E. coli enabled photophosphorylation when the bacteria were exposed to light (Martinez et al. 2007), and even generated a proton motive force, which turned the flagellar motor, yielding light-dependent motility (Walter et al. 2007).

Approach

some amazing text about how to approach a pizza

Cellulose production and secretion

Theory

Cellulose is a biopolymer produced by different species of gram-negative bacteria, especially by Acetobactors. An efficient producer of bacterial cellulose is Glucoacetobater xylinus, which produces large quantities of high quality cellulose. Cellulose is produced from the recourse glucose-6-phosphate. Glucose-6-phosphate is a key intermediate in the core carbon metabolism of bacteria given its importance in glycolysis, gluconeogenesis and pentose phosphate pathway. Even though the conversion of glucose and glucose-6-phosphate into cellulose is rather short, not only does the cell spends energy on forming UDP-glucose for cellulose production, it also uses glucose, which otherwise would have contributed to generation of ATP.
The ability for G. xylinus to produce, crystallise and secrete cellulose is controlled by genes in the cellulose synthase operon acsABCD. The acsABCD operon encodes four different proteins whereof AcsA and AcsB forms a dimer known as AcsAB. Together with AcsC, AcsAB is essential for biosynthesis of cellulose. AcsD has shown to be non-essential in production of cellulose, but absence of the protein results in decreased cellulose production, properly due to inappropriate crystallization of the polymer.
Other genera as E. coli excrete cellulose as a component of their biofilm. Even though cellulose production is intrinsic in E. coli, the in quantity the production is incomparable to cellulose production in G. xylinus. Indigenous E. coli is not capable of breaking down cellulose into a metabolisable energy source, but if the structural and water-holding polymer is enzymatically broken down into glucose residues, the cellulose polymer is a potent energy source. Read more here

Approach

After some research it was found that cellulose would be the perfect source of energy between the two bacteria in our project…. Imperial College London made the project Aqualose (link) in 2014, during which they produced excess amounts of bacterial cellulose.
The aim of this subpart of the project was to enhance cellulose production in the E. coli strain MG1655, to enhance the energy outcome of the entire system.
To enhance the cellulose production, parts containing the coding sequence of the Cellulose Synthase enzyme was transformed into E.coli strain MG1655. The original idea was to clone the entire cellulose synthase operon acsABCD under control of ptac. This was attempted many times, but with a total length over 9000bp the cloning was difficult, and unsuccessful. The plan was then to insert the cellulose synthase operon acsABCD into MG1655 on two plasmids, both controlled by the ptac promoter (http://parts.igem.org/Part:BBa_K864400). The catalytic domain, acsA, and the regulatory domain, acsB, both found in the part BBa_K1321334 (indsæt link), was attempted inserted into the vector psB1C3, carrying a chloramphenicol resistance cassette. Part K1321335 (indsæt link) containing acsC and acsD was inserted into the vector psB1A3, carrying an ampicillin resistance cassette. This was attempted many times, however, due to time constraints these clonings were also unsuccessful.
If the cloning of the cellulose synthase operon had been successful the cellulose production would have been tested using the fluorescent brightener 28... Read more here

Breakdown of Cellulose

Theory

Cellulose is a natural polycarbonate used in nature for a vast variety of biological purposes. It is most commonly found in plants, where it serves as the main structural component. Since plants are primary producers, it has caused many organisms of the Earth’s ecosystem to adapt accordingly Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews. 2002;66(3):506-77.. One of the key evolutionary features for the primary consumers was the development of the ability to degrade cellulose into glucose, which could then be used as a cellular fuel. A simple organism able to efficiently do so, is the Cellumonas fimi, which will convert cellulose to glucose in a two-step process with cellobiose as the intermediate Jung SK, Parisutham V, Jeong SH, Lee SK. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels. Journal of Biomedicine and Biotechnology. 2012;2012..

Breakdown of Cellulose to Cellobiose

Cellulose is a long polysaccharide consisting of β-1,4 linked D-glucose units. Many organisms, including E. coli, are incapable of producing enzymes able to deal with these strong β-linkages. To overcome this obstacle, the Cellumonas fimi developed to express two cellulases; the endo-β-1,4-glucanase (CenA) and the exo-β-1,4-glucanase (Cex) [kilde 7]. The endoglucanase is able to randomly break the amorphous structure of cellulose. This allows for the exoglucanase to break the β-1,4 linkages at every other D-glucose unit, thus releasing disaccharides in the form of cellobiose Lam TL, Wong RS, Wong WK. Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength. Enzyme and microbial technology. 1997;20(7):482-8.. Cellulose itself is practically too large to be transported across the cell membrane. This concludes that the breakdown of cellulose into cellobiose, must take place in the extracellular fluid.

The α-Hemolysin Transport System

The a-hemolysin transport system is an ABC transporter complex, consisting of the three proteins; TolC, hemolysin B (HlyB) and hemolysin D (HlyD) Gentschev I, Dietrich G, Goebel W. The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends in microbiology. 2002;10(1):39-45.. This ABC transporter complex effectively transports intracellular hemolysin A (HlyA) to the extracellular fluid. This system can be utilised to secrete HlyA tagged proteins, using a peptide linker to fusion the protein of interest with HlyA. Once a protein is HlyA tagged, it can be recognized by the ATP-binding cassette HlyB, which will initiate transportation of the HlyA tagged protein to the extracellular fluid. Gentschev I, Dietrich G, Goebel W. The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends in microbiology. 2002;10(1):39-45. Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microbial Cell Factories. 2012;11:8.

Uptake of Cellobiose

While cellulose is too large to be transported across the cell membrane, transportation of cellobiose is a common feature found in many organisms. An example is the E. coli, which utilises the membrane protein lactose permease (LacY) Sekar R, Shin HD, Chen R. Engineering Escherichia coli Cells for Cellobiose Assimilation through a Phosphorolytic Mechanism. Applied and Environmental Microbiology. 2012;78(5):1611-4.. Intracellular cellobiose can then be enzymatically broken down to glucose.

Breakdown of Cellobiose to Glucose

Through evolutionary events, many organisms have developed the ability to express enzymes capable of breaking the β-linkage of cellobiose. E. coli is known to express periplasmic β-glucosidase (BglX), which is known to have said feature by hydrolysing the cellobiose β-linkage[kilde 1]. The Saccharophagus degradans express an alternate enzyme, which can efficiently cleave the β-linkage in cellobiose. This is the so called cellobiose phosphorylase (Cep94A), which phosphorylates the cellobiose β-linkage resulting in its breakdown to D-glucose and α-D-glucose-1-phosphate Sekar R, Shin HD, Chen R. Engineering Escherichia coli Cells for Cellobiose Assimilation through a Phosphorolytic Mechanism. Applied and Environmental Microbiology. 2012;78(5):1611-4..

Approach

Cellulose to Cellobiose

In the pursue to make E. coli utilize cellulose as its only carbon source, we based our project on the Edinburgh 2008 iGEM team project, who developed two Biobricks containing the CenA and Cex genes. We then utilised the a-hemolysin transport system by creating HlyA tagged CenA and Cex, using a linker peptide. Once tagged with HlyA, the cellulases can be secreted to the extracellular fluid, where they actively cleave cellulose to cellobiose units. To implement this system in E. coli, it would require the heterogeneous expression of HlyB, HlyD, CenA-HlyA and Cex-HlyA.
To accomplish this we ordered DNA synthesis of CenA and Cex, each tagged with HlyA using a linker peptide. The genes encoding HlyB and HlyD was retrieved from the BBa_K1166002 by phusion PCR. Using the aforementioned parts, we composed the following construct for the degradation of cellulose to cellobiose:

Cellulose to Cellobiose

In the pursue to make E. coli utilize cellulose as its only carbon source, we based our project on the Edinburgh 2008 iGEM team project, who developed two Biobricks containing the CenA and Cex genes. We then utilised the a-hemolysin transport system by creating HlyA tagged CenA and Cex, using a linker peptide. Once tagged with HlyA, the cellulases can be secreted to the extracellular fluid, where they actively cleave cellulose to cellobiose units. To implement this system in E. coli, it would require the heterogeneous expression of HlyB, HlyD, CenA-HlyA and Cex-HlyA.
To accomplish this we ordered DNA synthesis of CenA and Cex, each tagged with HlyA using a linker peptide. The genes encoding HlyB and HlyD was retrieved from the BBa_K1166002 by phusion PCR. Using the aforementioned parts, we composed the following construct for the degradation of cellulose to cellobiose:

Optimised nanowires

Theory

Nanowires are produced by a small number of anaerobic bacteria. One of the well studied species is Geobacter sulfurreducens [4]. This metal and sulphur-reducing bacteria is easier to grow than other strains within the family of Geobacter bacterias. Naturally Geobacter lives anaerobically, but it does not fermentate. As a consequence of this, the bacteria has to oxidize the electron carriers in another way. Geobacter sulfurreducens utilizes an electrically conductive pili to transfer electrons to a metal and thereby reducing it. Reduction of Fe(III)oxide is the most occurring process in this organism. These electrically conductive pili makes up some extracellular filaments known as nanowires.
Geobacter Sulfurreducens is one of the most studied metal reducing bacteria and it was the first species of bacteria found to be capable of transferring electrons to metals to reduce them. The most common metal reduced is Fe(III)oxide. This process plays an important role in the geochemistry of soil and sediments [4]. The reducing-capability of Geobacter means that the bacteria is useful for cleaning waste containing heavy metals. Geobacter is a strictly anaerobic bacteria. The anaerobic environment is essential due to the reducing properties of oxygen, meaning that if oxygen was present it would steal the electrons thus hindering the electrons from being transferred to the metal through the extracellular electrically conductive pili.
The electrically conductive pili are composed of a type IV pili structural peptide PilA. Electron transfer in Geobacter is complex and beside the conductive pili the system consists of several c-type cytochromes responsible for the electron transfer to the terminal cytochrome oxidase. For example MacA c-type cytochromes are found in the inner cytoplasmic membrane and different Omc cytochromes are located in the outer cytoplasmic membrane [5]. The reduction of Fe(III)oxide by cytochromes in the periplasm and the production of electrically conductive pili. Studies are indicating that genomic regions up- and downstream from the pilA gene are essential for proper gene expression of the pilA gene [5]. Cloning the pilA gene into one of the prevalent E. coli laboratory strains and thereby making E. coli capable of express nanowires would with the current knowledge be infeasible. Different Geobacter strains encodes for different versions of the structural peptide PilA. Studies indicates that the content of aromatic residues in the structural pilin peptides are of great importance for the currents yielded. It has been presumed that the aromatic residues influences the structure of the peptide, thereby minimizing the intermolecular distance between the protein monomers [6]. Read more here

Approach

Our first idea was to either extract or synthesize the genes responsible for producing nanowires and insert them into E. coli as it is easier to work with. After a long period of researching different possibilities, we came to the same conclusion as the iGEM team from Bielefeld 2013, that the task simply is to comprehensive to be undertaken in the limited time frame we were under. Specifically you would have to take 16 kbp filled with illegal restriction sites and insert it in E. coli without any assurance it will work (get around 16 kbp into E.coli, have fun!). After consultation with post doc Oona Sneoyenbos-West we chose to work with optimising the nanowire producing bacteria, Geobacter sulfurreducens.
The genus Geobacter contains bacteria capable of producing conductive pili, which in our case can be utilized to transfer electrons to an anode. This way we can harness the energy produced and use for charging smartphones or powering or products. But we are not just going to use the regular Geobacter sulfurreducens strain, no we are going to optimise it by exchanging its pilA gene with the pilA gene from G. metallireducens. The first step was to have pilA from G. metallireducens synthesized in two pieces as IDT DNA’s gBlock system will not allow repeats. The two fragments were assembled and inserted into the pSB1C3 backbone (Here we can insert part number etc.). As we wanted to insert both pilA and the chloramphenicol resistance cassette from the backbone into G. sulfurreducens chromosome, phusion PCR was used to create a PCR product that contained pilA and chloramphenicol resistance. The upstream gene XapR and the downstream gene pilR are both important in the expression of the pilA gene, and we want to make a recombination by only changing the pilA gene from Sulfurreducens with the one from metallireducens. Therefore a small part of XapR and pilR, 500 bp, were made to PCR cassettes. The two PCR fragments were made from the genome extraction of Geobacter sulfurreducens. All PCR product contained overhangs from the primers. The two overhangs on XapR and pilR were linked with the pilA-chloramphenicol brick through several phusion PCR and the finished insert was ready: 500 bp PilR - chloramphenicol - pilA - 500 cp XapR.
To get the PCR product into the Geobacter Sulfurreducens PCA an electroporation was carried out (SOP26, skal bare være link). After the night the Geobacter was transfered to a media containing chloramfinicol with a concentration of 10 µg/ml. Which is the concentration that makes it possible to select for the acquisition of chloramfinicol resistant Geobacter (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92998/) .
For testing the nanowires an ??? experiment was executed. Here BBa_K2449002 in Geobacter Sulfurreducens was tested up against a Geobacter Sulfurreducens wild type, E. Coli wild type and E. Coli containing the outer membrane porin brick BBa_K1172502, from Bielfeld-Germany 2013´s iGEM project. BBa_K1172502 was introduced in the testing to compare the nanowires to a previously successful energy producing brick in the iGEM competition. The best brick contains the T7 promoter, which means we need a strain that have the gene for the T7 RNA polymerase. In our lab we used E.coli strain ER25663127. The polymerase is controlled with a LacZ gene. This way the polymerase can be activated by adding IPTG, by this way activating our target gene inderectrly. The M9 medium is the same as they used to test with at bielefeld (https://2013.igem.org/Team:Bielefeld-Germany/Labjournal/ProtocolsPrograms#M9_Medium), it is only the setup that is a bit different. The setup is the same as the other two flasks with Geobacter. Read more here

Modelling

In order to find the best way to implement the toxin-antitoxin system, we resort to modelling. We use the gillespie algorithm to model the interactions of the toxin antitoxin system.
We find that when we implement enhanced relE production as a tool to make the bacteria dormant, an additional implementation of relB to ensure don’t stay dormant when in light again.
The model found that the system is sensitive to the relE:relB ratio as well as the total production, and that an implementation with production rates in the vicinity of 50 and 35 molecules pr. min for relB and relE respectively yields close to the wished for effect: THe bacteria goes dormant in an hour and wakes up quickly.

Approach

We are modelling the RelE RelB toxin-antitoxin system. RelE is a toxin restricting growth, by inducing a dormant state. This is inhibited by RelB, which forms complexes with RelE. Two different complexes are made: RelB₂RelE and RelB₂RelE₂, containing 1 and 2 RelE molecules respectively.
Both RelE and RelB are expressed from the same promoter, RelBE. When only small amounts of RelE is present, RelB and RelB₂RelE represses transcription of RelBE, by binding to the operator.
At higher concentrations of RelE, the toxin mitigates this repression, by reacting with bound complexes.

For natural purposes the half life of RelB decreases significantly under starvation due to lon-protease, with shifts the equilibrium of RelB and RelE to a high state of RelE. The interactions with the promoter, keeps the amount of free RelE at a very low value outside starvation and stabilises the system. In our simulation the shift in equilibrium is made by introducing additional translation of RelE.
We used two model in two ways. First we saw how a given configuration of relB and RelE production increased the relE concentration and if it could cause dormancy within 2 hours. Second we investigated for how long each configuration

Rates and reactions

In the units off all reaction rates we use the approximation that in an E.coli. with a size of 1-2 μm, 1 molecule in the cell = 1nm. Thus we convert all units to be measured in molecules, as this fits the premises of the gillespie algorithm.
The RelB forms dimers at a high rate, so we assume all present RelB to be in dimers, capable of forming complexes with RelE.
RelB has a relatively low half-life at about 3-5 minutes, while RelE is stable and it’s half life is an effect from dilution due to growing bacteria (we use 43 min). During dormancy, growth is restricted and we increase RelE half life to 2000 min (around a day) as the dilution.
The transcription rates of RelE and RelB is based on the concentration of RelE and RelB under stable conditions. Here RelB is 10 times more prevalent than RelE (citation 2), so to make up for the higher half life of RelE, RelB has a much higher transcription rate than RelE (100 times)
The complexes are close to stable and given the same half life as RelE. However, to get free RelE to work RelB in complexes need to decay as well. The rate is set to a fourth of free RelB.

For the promoter bindings we let the operator be inhibited by binding with either RelB or 1-2 RelB₂RelE, given that the operator has to binding sites. We consider the cell to have four chromosomes with one promoter on each (less chromosomes would let the system work, but with more noise).
The initial values in the model are given by:

insert table with numbers and constants

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.
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 only need a production of 0.01-2 (assuming a few hundred copies).
Considering the inherent toxin-antoxin system activating under starvation, we see that the the magnitude of relE copies around 50-80 molecules pr. cell. This makes it reasonable to believe that the cell enters dormancy when a few tens of free relE copies.

Running the model

The model is using the gillespie algorithm to give a stochastic view of the system and is run on matlab. The code uses an implementation by matlab user Nezar (https://se.mathworks.com/matlabcentral/fileexchange/34707-gillespie-stochastic-simulation-algorithm).
For the dormancy runs we input deterministic initial values, and then let the system run 30 min without activating the inserted toxin promoter. This results in a stochastic distribution of initial values mimicking variations between cells. Analysis show that 30 minutes is enough for the model to find a stable distribution, which is realistic considering the growth cycle of an e.coli.
For wakeup runs, we use the data generated at the end of a sleep run as initial value and remove the production of relE. We consider a cell to be woken up when the concentration of free relE decreases below 15 copies. This is perhaps a low value, but tests show marginal difference between 15 and 45 copies, where the low bound 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. The model has no cut off for maximum values of relE, as we don’t know the exact relation between relE concentration and hibernation state, yet as a functional cutoff is found through wake-up times, it is not necessary.

Experiments

lol, what is this?

Demonstration and Results

results? never heard of it

Parts & Procedures

In this section, you will find all the needed information to replicate our approach and experiments. Both the parts, notebook, SOPs and protocols will show in a pop-window, from which you can obtain all the needed information, should you be interested. An essential part of going to the lab is risk and safety assessments. This you will find at the end of the section, so just go ahead and keep on scrolling.

Parts

BBa_K2449000
Contain the genes for pilA-N found in G. Metallireducens.

BBa_K2449001
Contain the sequence for the PilA-C genes of G. Metallireducens.

BBa_K2449003
Contain the sequence for the Cep94A gene, an enzyme that breaks down cellobiose.

BBa_K2449008
Contain the sequence for the antitoxin RelB.

BBa_K2449010
Codes for a Ptac promoter regulating csoS2.

BBa_K2449011
Contain the sequence for CenA (Endoglucanase), encoding a cellulose degrading enzyme. It was optimised for E. coli.

BBa_K2449012
Contain the sequence for Cex (Exoglucanase), encoding a cellulose degrading enzyme. It was codon optimised for E. Coli.

BBa_K2449013
Contain the sequence for CenA (Endoglucanase) fusioned with HlyA using a G-Linker. Optimised for E. Coli.

BBa_K2449014
Contain the sequence for Cex (Exoglucanase) codon, together with G-Linker and HlyA.

BBa_K2449015
Contain the sequence for HlyB + HlyD, a system used for secretion of HlyA.

BBa_K2449002
Contain a LacI promoter, RBS, pilA-C from G. Metallireducens, RBS, PilA-N from G. Metallireducens and a double terminator.

BBa_K2449004
Contain a LacI promoter, RBS, Cep94A and a double terminator.

BBa_K2449005
Contain a LacI promoter, RBS, BglX and a double terminator.

BBa_K2449006
Contain a promoter, RBS, BglX and a double terminator.

BBa_K2449009
Contain a LacI promoter, RBS, the gene sequence for RelB, controlled by a Lacl regulated lambda pL hybrid promoter and a double terminator.

BBa_K2449016
Contain a PenI promoter, HlyA tagged CenA.

BBa_K2449017
Contain PenI promoter, HlyA tagged Cex and a double terminator.

BBa_K2449018
Contain PenI promoter, HlyB, and HlyD.

BBa_K2449019
Contain HlyA tagged Cex, PenI promoter, HlyB, and HlyD.

BBa_K2449020
Contain a PenI promoter, HlyA tagged CenA, PenI promoter, HlyB, and HlyD.

BBa_K2449021
Contain a PenI promoter, HlyA tagged Cex, PenI promoter, double terminator, HlyB and HlyD.

BBa_K2449022
Contain a promoter, HlyA tagged Cex, HlyA tagged CenA, a double terminator, a promoter, HlyB and HlyD.

BBa_K2449023
Contain a PenI promoter, HlyB and HylD.

Notebook

SOPs and Protocols

SOPs

Protocols

SOP01 - LA plats 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

Safety

Proper Risk Management

Biosafety and proper risk assessment are important aspects to consider before any handling of genetically modified organisms (GMOs). There are several concerns that must be properly addressed. The safety of the public as well as of the environment, is of the utmost importance, but the safety of the person in direct contact with the GMOs shouldn’t be compromised either. The risk associated with laboratorial work can be evaluated using the statement “Risk = Hazard x Probability” (link). To responsibly assess this inquiry, the entire team was given a mandatory lab safety course held by Lab Technician Simon Rose. In addition, we received a detailed handbook regarding lab safety. This ensured that all our team members were well equipped to work safely in the lab at all times. Throughout the project we have continuously been evaluating the safety of our work. These assessments can be found in the safety form (link).
Furthermore, our team participated in the 5th annual BioBrick workshop hosted by DTU BioBuilders. Here we engaged in a lab safety course before entering their lab. Both of these lab safety courses gave us the necessary knowledge to work safely with GMO, proper handling of waste and the according procedures in case of an emergency.
In the lab, we worked with several potentially harmful chemical agents such as DMSO (dimethyl sulfoxide), ethidium bromide, chloroform, phenol, Congo red, antibiotics and autoclaved glycerol. These chemical agents were handled using gloves at all times, and, whenever deemed necessary, handled in a fume hood. We used a UV board to visualize bands in agarose gels. UV rays are carcinogenic when exposed frequently and for longer periods of time. To reduce the amount of exposure, several precautions were made; gloves, long sleeves and a facial screen were worn at all times and the time spend at the UV board was no longer than the necessary. GMOs were always handled wearing gloves, and all team members wore clean lab coats restricted to the laboratorial areas.

Public and Environmental Risk Assessment

The chassis organisms containing the system is meant to be contained within a container, which should be incorporated into an urban environment. While this device would be a safely enclosed container, it still possess the risk of physical breakage from violent acts or environmental disturbances. It is for this reason, that we consulted a plastics expert, who advised us to use the plastic known as Polycarbonate. This plastic is remarkably durable, with the ability to ward off most physical traumas. As such the plastics expert believe that the container would last in an urban environment for at least 20 years, and most likely more than that. To illustrate the durability of the plastics, he notified us of several devices from the 1970ies made of the same plastic, that still stand strong today.
One of the biggest concerns would be the release of GMOs into nature. While the GMOs used aren’t pathogenetic, they would be able to share the plasmids containing antibiotic resistance selectors to other pathogenic bacteria. Antibiotic resistance in pathogenic bacteria, complicates the treatment of an infected individual, and could in tragic cases be the line between life and death. However small this scenario is, it should be addressed properly. Furthermore, antibiotic resistant E. Coli strains could outmatch some of their fellow E. Coli strains through natural selection. This could negatively affect the natural balance, that we are aiming to restore with the development of the PowerLeaf.
To safely avoid these risks, there should be implemented several kill switch mechanisms into the final device. This could be performed by implementation of a light sensing system into the energy converting unit, which would turn on the kill switch if exposed to light. This would of course mean, that the energy converting unit’s container, would need to block all sunlight. A task that could easily be carried out by adding Carbon Black to the required areas of the container. The energy storing unit, which requires light to actively function, could then have a kill switch which makes it completely dependant on presence of the container. This could be accomplished by having harmless molecules not naturally found in nature circulate in the system. Which should be required for the survival of the energy converting unit. A similar effect could be accomplished by making the energy converting and the energy storing units codependent on each other for their survival. The implementation of such kill switch mechanisms, would tremendously improve the biosafety of the device, by opposing hazards related to any kind of physical breakage.

List of Assessed Items

Chassis Organisms
Escherichia coli strains: K12, TOP10, MG1655, KG22, BW25113, ER25663127
Geobacter Sulfurreducens strain: PCA

Vectors
pSB1A2: An iGEM plasmid backbone carrying an ampicillin resistance gene
pSB1C3: An iGEM plasmid backbone carrying a chloramphenicol resistance gene
pSB1K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB3K3: An iGEM plasmid backbone carrying a kanamycin resistance gene
pSB4K5: An iGEM plasmid backbone carrying a kanamycin resistance gene

Bacteriophages
P1 phage, using its site-specific recombinase for transduction of E. Coli

Practices

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Vestibulum tincidunt ac nisl at mattis. Sed eu mollis nisi. In pulvinar mi velit, dictum congue sapien ornare vel. Integer euismod varius velit ac euismod. Curabitur dapibus eget neque hendrerit sollicitudin. Etiam nec consequat diam, interdum egestas purus. Nullam ultricies et augue at vestibulum. Proin ac velit ac nibh rutrum varius at id metus. Morbi vitae auctor arcu, eget pulvinar mi. Suspendisse potenti. Fusce ornare nisi a volutpat malesuada. Donec sed augue nisl. Vivamus et dui orci. Suspendisse potenti. Ut luctus, nisl in ullamcorper facilisis, purus tortor eleifend odio, nec efficitur erat nisl vel massa. Suspendisse sed velit molestie, tincidunt nulla in, consectetur ligula.

Bioethics

ethics is forcing Neergaard drink phenol

Jonas can approve on this

Integrated Practices

“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.

integrate a pizza here

A Statement from the Mayor of Odense

We first decided to reach out to the mayor of Odense, to investigate the possibilities for iGEM to help in the government's endeavours to make Odense a CO₂ neutral city, with a high quality of life.

“We face a series of challenges that we have to recognize, in the chase of a good and sustainable life in the city of Odense. Some of these concern local circumstances, while others contain national and even global issues. We as the municipality can only go so far on our own. So we are entirely dependant on the help of local agents. It makes me so happy, when the students of the city, have taken on the mantle of developing new green technologies, which global issues while also contributing to the city's high quality of life.”

Peter Rahbæk Juel - Mayor of Odense

The core philosophy of our integrated human practices has been to integrate local experts in the development of our project; in order to better comprehend how to use the knowledge gained in the laboratory to shape a product that would compliment Odenses (or beyond) green values. We have also made use of experts in other fields in order to better understand how to shape our project - and so our human practice has influenced everything from the design of our prototype(s), laboratory work to ethical considerations.
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.

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.

From Mrs. Dienhart’s point of view, one of the most advantageous attributes of our device, is the the potential for changeability in the size and shape of the PowerLeaf - as this means it could be shaped depending on what urban area we wish to integrate the PowerLeaf within. We had yet to consider the PowerLeaf as a device not limited by physical dimensions, and it’s perhaps the most significant element we took away from our meeting with Mrs. Dienhart. Changeability is a necessity to a city planner, as various laws and aesthetic aspects need to be taken into consideration, when altering or creating an urban environment.
Accessibility – the citizen will not use our device if it is not easily accessible. This means that the overall design of PowerLeaf – regardless of its aesthetic – always needs to be designed with a user in mind. Offering a mobile-charger in a city-space is only clever insofar that the citizen using the public space is aware of the device and how to easily access it. Reflecting on the advice of Mrs. Dienhart, we decided to reevaluate the means of implementation of our bacterial solar battery in the prototype, to ensure that the need for accessibility and user-comfort is met.
Mrs. Dienhart supported our notion that offering free and accessible energy within public space could help ensure that the ordinary citizen of Odense uses and stays in the public space for a longer amount of time. Something that is valuable not only to the individual citizen but also to the community as a whole, as it creates a sense of city-cohesion and hence a high quality of city-life.
Overall Mrs. Dienhart confirmed that our PowerLeaf could play a part in Odenses dream of developing into an even greener and more lively city. She also made us aware that not every neighbourhood in Odense will be desiring the same design, and that we ought to focus on the changeability aspect in the development of our prototype’s design.

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.
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.

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.
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.

“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
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:

Solar exposure - The material covering the solar cell, must allow sunlight to pass through it to the bacteria.
UV resistance - As the material will be exposed to the sun, it should be resistant to the UV radiation.
Bacterial growth - The material must support, or not be toxic to the bacteria.
Easy to mold - As the device is only dependant on the insides, the outside could be molded depending on the co
Durability - material must be able to withstand hard conditions and heavy weight.
Temperature -The material must allow for appropriate temperature for the bacteria, despite the constant sun exposure.
Longevity - We would like for the material to have as long a durability as possible, as replacing the leaves often would prove cumbersome. In this regard we are aiming for at least twenty years.
Price - We are looking for a material that is as cheap as possible, without sacrificing the necessary criteria.
Environmentally friendly - Considering the goal of this project being the creation of an environmentally friendly energy source, the ideal material would be as environmentally friendly as possible.

Interview with Flemming Christiansen
For the purpose of finding the necessary materials for our prototype, we contacted one of the leading plastic experts in Denmark, Flemming Christiansen, who acts as the market development manager of SP Moulding. He has been acting as a plastics consultant, since his graduation as a master of science in Engineering with a speciality in plastics 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.

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.
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

hello my friend, wanna learn something? the wiki isn't done yet.

lets goo

Prospects

Our prospects section is aimed to expand on our visions regarding the PowerLeaf. A vision we would very much love to see become a reality. For this reason, we have concentrated on creating an overview of the project, for the benefit of future iGEM teams. Hopefully it can assist prospective teams on how to take the PowerLeaf to the next level. Lastly, we decided to list some of our project ideas to the teams wishing to create a completely new project. Perhaps some of these ideas can be used by prospective iGEM teams, or just help to kickstart their creative thinking.

Perspectives

Building a Product for a Better Future

The purpose of the PowerLeaf, is to provide a greener alternative to the currently available energy sources. An important aspect of such an undertaking, is to limit the use of depleting resources, such as silicon, in the construction of the device itself. This is accomplished through the use of the most common resources available. This will contribute to our dream of building a better future, where fear of reaching a critical shortage of natural resources has been eliminated. The production of the PowerLeaf itself is made easier too, as the device benefits from the bacteria's ability to self-replicate, if provided with essential nutrients.
As tools for genomic editing improves, the advancement of biological devices will conceivably become even more complex and independant. They will do so by introducing new metabolic pathways inspired from other organisms using genetic engineering. This could potentially allow the PowerLeaf to become completely independent through its self-replication, by producing their own essential nutrients directly from unwanted pollution in the environment. A process that would lead to cleaner cities, along with providing a natural solution to sustainable energy.
The PowerLeaf will be representing a natural leaf design, thus leading to a nature-in-city ambiance, which can have a soothing effect in the ever so stressful cities. Not only will the design represent a plant leaf, but some of the key functionality aspects of the device are inspired from those of a plant leaf. Hereby, we refer to photo synthesis and building cellulose as a biological product.

Genetic Code Expansions for Biological Engineering

Expanding beyond those technologies used in today's Synthetic Biology, many research groups are working on genetic code expansion. We had an interesting talk from post.doc Julius Fredens, about his work on genetic code expansion. Once a technology like this advances, it will completely revolutionize biological engineering, including that of the PowerLeaf. Genetic code expansion could be used for optimization of the systems in the PowerLeaf; optimization of nanowires, improvement of the light-sensing system and making the breakdown of cellulose inducible.

To Future iGEM Teams

Hello future iGEM’er and welcome to the section where you are the center of attention. First of all, congratulations on starting your iGEM journey, you are going to have a amazing summer with plenty of wonderful experiences and new friendships. In this section, there will be two main topics, improvement and further development of our project, the PowerLeaf, and some of our project ideas generated in the startup phase to use for your project or start your creative thinking.

Further Development of Our Project

For those of you, that found interest in our project this year and would like to continue on improving it; this is the section you were looking for. We have listed the systems and the related information on theses needed for the device we envisioned, however you should not be limited to those. You are more than welcome to contact any of us regarding questions to the project, you can find our email addresses to each of us in the Team section of the Credits.

Systems that did work:

Light sensing system, this is used by the energy storing unit to reduce metabolism during times of the day with low amounts of solar energy available for the energy production, i.e. night time. We had several failed attempts during the development and optimization of the system and have through this learned a lot about the system. Furthermore, the system was modelled to gain an even greater understanding about the regulation of its light sensitivity. You can read about the work we did regarding the light sensing system here.

Cellulose consumption, this was used by the energy converting unit to degrade cellulose to glucose from which electrons could be retrieved. This system is probably the most straightforward, but was also worked on very extensively. You can read about the work we did regarding cellulases here.

Optimisation of the nanowires, this system was heavily inspired by the following article (link). We did create the required BioBricks to make the system work, but still requires some extensive work to actually implement it. You can read more about the work regarding the nanowires here.

Systems that didn’t work:

CO₂ fixation, we retrieved the parts from the Bielefeld 2014 iGEM team and worked on assembling their parts into one fully functional BioBrick. However, we had a lot of trouble assembling it, and it seems that Bielefeld 2014 didn’t succeed on combining all the components needed for CO₂ fixation either. So be aware of this. It seems like a simple assembly, but has caused us lots of problems. Some of the larger BioBricks tend to do that when they reach a certain size. You can give it a go anyways, but make sure to have a backup-plan, or maybe even try to redo the CO₂ fixation by using a system from a different organism. We essentially decided to let go of this system of the PowerLeaf to focus on some of the other components. You can still read about our work done regarding the CO₂ fixation here.

Cellulose production and secretion from the fixated CO₂. These parts were retrieved from the Imperial College London 2014 iGEM team, this, much like the CO₂ fixation, gave us trouble when it came to the assembly of the large BioBricks. It did seem that Imperial College London 2014 made their system work, but in the end, they proved it to be very inefficient of producing cellulose. So, this part could be the very thing to improve. You can still read about our work regarding the cellulose production and secretion here.

Systems we didn’t work on, but should be implemented in the device:

ATP production from solar energy comes to mind as one of the most essential system needed for the PowerLeaf to actually work. We had to pick the some of the systems to work on, and at the end of the day, this was the project our supervisors recommended to cut, if we wanted to work on more than just one system. Instead, we had a great Skype call early in the project with the Australian Macquarie iGEM team, whom has been working for many years with the implementation of the photosynthetic systems in E. coli. You could always contact them regarding the photosynthetic systems, they are super nice.

Making the interaction between the cellulose and the cellulases a controllable element, so it could be controlled in the same way of an on/off switch. This is also a very crucial part of the PowerLeaf, since it would otherwise be generating an electrical current non-stop. Even when it is not needed, and thereby overthrow the potential for long term-storage of solar energy. We believe this can be solved either through precise gene circuit regulation or by physical compartmentalization, however there might be even more elegant ways to solve this issue.

Physical engineering of the hardware required to make the device work. It should be possible for the energy storing unit to convert CO₂ to cellulose, which will produce O₂, thus making its chamber aerobic. For the energy converting unit to effectively transfer retrieved electrons to an anode, it will need to be in an anaerobic chamber. This will be a very difficult obstacle to overcome and requires some out-of-the-box thinking, to come up with a novel idea without having to require more energy than produced by the system. Engineering of the hardware required, e.g. anode, chamber, circulation of important nutrients and use of the correct plastic, is really important to make a workable prototype of the PowerLeaf. We worked out the optimal type of plastic for the system with the help of local experts. You can read about our work regarding the plastic here.

Ideas from Our Idea Generation

List of ideas from our idea generation

Credits

Just like in movies, you get to meet the brilliant minds behind the project in the credits. Some might leave the cinema without reading the credits, but we hope you will continue to read ours, as it’s just as important and you will get to know us on a more personal level through this. We probably have more in common than you think. Behind every great team is a great amount of external attributions. The contributors have supported and inspired us, especially when things have been rough and deadlines near. Afterwards you can turn your attention to our collaborations, which was an amazing experience, this really shows of the true iGEM spirit.
Last but not least, don’t miss out on the ‘after-the-credits-clip’, which summarizes the fun we had during this wonderful iGEM experience. This is especially important, since you get the words ‘thank you for listening, we hope you enjoyed our wiki and project’ - we know you have been waiting impatiently to reach that part of the wiki.

Team

Welcome to the team page, here you get to know us on a more personal level. As a team, we are 12 students from 8 different majors. As friends, we experienced the most amazing summer together, filled with various fun activities, both in- and outside the lab. To mention a few; we had road trips, dinners, Game of Thrones night and we even celebrated Christmas in July! We shared all of this with our amazing supervisors, for which we are truly greatful.

Emil Bøgh Hansen

Study: Biology
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.

Emil Søndergaard

Study: History
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.

Emil Vyff Jørgensen

Study: Physics
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.

Ellen Gammelmark

Study: Biochemistry and Molecular Biology
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!

Felix Boel Pedersen

Study: Biochemistry and Molecular Biology
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.

Frederik Bartholdy Flensmark Neergaard

Study: Biochemistry and Molecular Biology
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.

Frederik Mark Højsager

Study: Medicine
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.

Jonas Borregaard Eriksen

Study: Pharmacy
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.

Lene Vest Munk Thomsen

Study: Philosophy
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.

Malte Skovsager Andersen

Study: Biochemistry and Molecular Biology
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.

Sarah Hyllekvist Jørgensen

Study: Biochemistry and Molecular Biology
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.

Sofie Mozart Mortensen

Study: Biomedicine
E-mail: sofmo15@student.sdu.dk
Hi there! My name is Sofie, and I am the team mama! I am the one who makes sure everyone gets their fair share of cake. When I’m not in the kitchen, busy making cakes for my teammates, you can find me in the lab, where I’m working on enhancing our systems cellulose production.

Attributions

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:

Assistant professor Mikkel Girke Jørgensen, for his general support and advice on the project, the laboratory, the fundraising and our team synergy.
Ph.D. student and former iGEM participant Patrick Rosendahl Andreassen, for his guidance and technical assistance in the laboratory.
Ph.D student and former iGEM participant Thøger Jensen Krogh, for his help in developing the wiki, as well as his laboratory guidance.
Cand.phil student and former iGEM participant Tim Munk, for his focus on team dynamics and advice for our human practices.

We would also like to thank:

Academic assistant Tina Kronborg, for her guidance in the lab, as well as for providing us with lab equipment.
Medical Laboratory Technician Simon Rose, for giving us a course in lab safety, risk assessment and general guidance in the lab.
Postdoc Oona Sneoyenbos-West, for providing us with Geobacter Sulfurreducens PCA and the necessary knowledge on how to grow this particular bacterial strain. Furthermore, she helped us greatly with helpful discussions regarding the advancement of our project. We would also like to thank her for lending us her laboratory, for the cultivation of Geobacter Sulfurreducens PCA.
Postdoc Satoshi Kawaichi, for his assistance in measuring the electrical conductivity of our nanowires, as well as providing us with knowledge on the Geobacter Sulfurreducens.
Business scout and PhD Ann Zahle Andersen, for presenting us with the necessary tools for the development of innovative business ideas.
Stud.scient Kristian Severin Rasmussen, for helping us use the oCelleScope for testing.
Stud.scient Brian Baltzar, for hosting a workshop regarding Adobe Illustrator, which has been a great help to the development of graphics for our wiki.
Ph.D student Richard Xavier Etienne Valli, for helpful discussions in the lab.
Software Developer Jonas Hartwig, for his help with some JQuery functionality on the wiki.
Stud.scient Birka Jensen, for general advice and suggestion on how to build an iGEM wiki.
Stud.med Ida Charlotte Hvam, for helpful discussions on the development of our wiki, helping with last minute figures to the wiki, as well as proof-reading of its content.
Ph.D student and current iGEM advisor of the team from Bielefeld, Boas Pucker, for providing us with BioBricks created by former iGEM teams from Bielefeld.
Our iGEM HQ Representative, Traci Haddock-Angelli, for her general guidance and assistance in registering our meetup to the official iGEM meetup page.
iGEM HQ Representative and Lab Technician, Abigail Sison, for her help in registering our meetup to the official iGEM meetup page.
Stud.polyt Oliver Klinggaard, for helpful discussions on the implementation of a pan-tilt system and for providing os with his project report on the subject.
DTU BioBuilders, for hosting their 5th Annual Biobrick Workshop. And for attending our meetup.
The UNIK Copenhagen iGEM team, for hosting the Nordic Meetup. And for attending our meetup.
The TU-Delft iGEM team, for hosting the European Meetup.
Mimo Antabi, for adding our adverts to the university info-screens preceding the Danish Research Festival.
Allan Haurballe Madsen, for helping us with our appearance at the Danish Science Festival.
Outreach Coordinator and PhD Lise Junker Nielsen, for for helping us with the Danish Science Festival as well as with the visit from the Academy for Talented Youth. We would also like to thank her for providing us with iPads for laboratory use.
The Danish Science Festival, for having us at their annual event. We would also like to thank all the visitors who attended our booth.
The high schools Odense Technical gymnasium, Mulernes Legatskole and Academy for Talented Youth, for letting us present our project.
The UNF Biotech Camp, for having us present our project to the attending students.
The elementary school, Odense Friskole, for letting us present our project for their 8th grade students.
All former iGEM participants from SDU, for attending our preliminary presentation and giving us feedback before the Giant Jamboree.
The following groups and associations, for helping us develop our human practices: SP-Moulding, Borgernes Hus, Kommunens bygninger, Bolbro - områdefornyelse, Odense Byudvikling.
Matlab user Nezar, for an easy implementation of the gillespie algorithm into matlab.

Sponsors

Thanks to:

The Faculty of Science at University Southern Denmark, for providing us with the fundamental funds required for our participation in the iGEM competition, and for providing us with lab benches and essential equipment.
The Faculty of Health Sciences at University of Southern Denmark, for their much needed funding of our project.
Integrated DNA Technologies, for providing us with 20 kilobases of gBlock gene fragments.
SnapGene, for providing our team with memberships to their software during the duration of the competition.
PentaBase, for sponsoring us with 10.000 DKK worth of oligos and a further 10% discount.
Eurofins Genomics, for providing us with an 80% discount on a Mix2Seq kit.
CO₂NeutralWebsite, for attributing to green energy in our name, and thereby eliminating the carbon footprint our wiki makes.
Piktochart, for extending their student-offer to our mail aswell, providing us with easy access to great graphics.

Litterature

Project Synergism

We have all been working together in every aspect of our project. Nevertheless, some people have had to focus on some areas more than others. The main groups are listed as follows;

The group focusing on fixation of CO₂, production of cellulose and light-sensing dormancy consisted of Sarah Hyllekvist Jørgensen, Ellen Gammelmark, Sofie Mozart Mortensen and Emil Bøgh Hansen.
The group focusing on the breakdown of cellulose to create an electrical current and optimisation of nanowires consisted of Felix Boel Pedersen, Frederik Bartholdy Flensmark Neergaard, Jonas Borregaard Eriksen and Malte Skovsager Andersen.
The group focusing on the implementation of the device in an urban environment, as well as our outreach consisted of Emil Søndergaard, Frederik Mark Højsager and Lene Vest Munk Thomsen.
The mathematical modelling of our project was single-handedly performed by Emil Vyff Jørgensen.
Coding and design of the wiki was performed by Felix Boel Pedersen and Frederik Mark Højsager.

Collaboration

"Alone we can do so little; together we can do so much"

The American author Helen Keller had it right! As an iGEM team, you can reach many goals, but as an entire community, we can aspire to achieve so much more. Thanks to all the people that made this iGEM experience so memorable, we truly enjoyed your companionship!

Danish ethics and wiki workshop at SDU

In the spirit of the iGEM community, we hosted a meetup in August for our fellow Danish iGEM teams: InCell from the University of Copenhagen (KU), and the Snakebite Detectives from the Technical University of Denmark (DTU). A total of seven members from these two teams joined us for breakfast and attended our meetup. This was the first ever iGEM meetup hosted by our university, so we decided to make it memorable. We decided to take advantage of our interdisciplinary team roster, and designed a wiki and ethics workshop to aid our fellow Danish teams.
We utilised the broad interdisciplinary profile of our team, to have Emil S. and Lene present the perception of science throughout the history and the bioethical aspects of GMO, respectively. Emil S. has a Bachelor of Arts in History, and Lene has a Bachelor of Arts in Philosophy. The ethical presentation was purposely turned into an ethical debate, where viewpoints of ethical conduct were exchanged and discussed. After the presentations and discussions on bioethics, it was time for the wiki workshop.
The SDU-Denmark iGEM teams have won the Best Wiki prize several times in the past. As such, we wanted to share the knowledge gained from our university's past. To facilitate this exchange of knowledge on wiki development, we recruited our current supervisor Thøger Jensen Krogh, to hold presentations on how to design a good wiki. He was qualified for this task through his role as the designer of the SDU iGEM 2013 and 2014 team wikis, which won the special prize on both occasions. During the presentation, Thøger had arranged several exercises where the attendees got to mingle, discuss and evaluate their wikis. This resulted in a steady flow of information and constructive feedback between all three teams.
After a long day of learning and discussing, we went for a tour around campus under the summer sun, which concluded in a visit to the roof terrace of the campus dormitory, followed by dinner. It was requested, by our fellow Danish teams, to make the SDU meetup a tradition. They suggested for all of us to meet again closer to the wiki deadline, to evaluate each team’s progress.

Attending meetups

Besides hosting our own meetup, we also attended several ones during our iGEM experience. The first of which, was the 5th Annual Biobrick Workshop in March, hosted by the Technical University of Denmark. This meetup not only gave us our first experience with Biobricks, but also worked as a foundation for friendships across the teams.
Our second meetup, the Nordic iGEM Conference, was hosted by the University of Copenhagen in June. The main focus of this meetup, was the traditional mini Jamboree. Participating in this gave us useful feedback from the judges, as well as from our fellow iGEM teams. This helped us greatly shape and develop our project for the better.
To celebrate the beginning of our iGEM summer, we went on a road trip to attend the European Meetup, hosted by the Delft University of Technology in the Netherlands. Here we discussed ideas regarding our project at a poster session, learned from all the other great iGEM projects, and made new friends from all over Europe.

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.
As our project revolves around global warming and green sustainable energy, we were thrilled to hear about the iGEM Goes Green initiative from the TU Dresden iGEM team. Following their guidelines, we have calculated the carbon footprint of our laboratory work and travelling. We have, in part, tried to make up for our carbon footprint, by changing our travelling and eating habits in our everyday lives. Furthermore, we have reduced our daily electricity consumption, our wiki became CO₂ neutral and we made an effort to sort our waste. The full report can be scrutinized here.
We sought expertise from the Macquarie iGEM team, who has worked with the implementation of photosynthesis in E. coli since 2013. We had an interesting Skype call with their team, where we discussed the particular challenges the previous teams had experienced throughout their projects. During the skype conversation, we realised, that they could benefit from our knowledge on the electron transport pathways, that we used for our project.
We were also able to help the Stony Brook iGEM team by facilitating communication with members of the SDU iGEM team from 2016. Shortly after the European meetup, we received an email from the Cologne-Düsseldorf iGEM team regarding a postcard campaign, which we gave some feedback on.
During our project we received several questionnaires from fellow teams. We were delighted to help the teams by answering their questionnaires. The questionnaires were from:

Waterloo - regarding 3D printing of lab equipment
Dalhousie - regarding the common conception of science literature
University of Washington - regarding communication platforms used by teams
Vilnius-Lithuania - regarding cotransformation
Nanjing-China - regarding whole-cell sensor for formaldehyde
University of Sydney - regarding the use and accessibility of insulin
Georgia State - regarding disabilities
Greece - regarding modular RNAi-based logic circuits

Final Words

‘Thank you for your time, we hope you enjoyed our wiki and project’. Now you can sit back, relax, and be proud of your hard work. While you do so, feel free to enjoy some of the less serious pictures and snippets of stories from our amazing iGEM summer.

lots of fun stories and pictures

All the best,
SDU-Denmark 2017

@@ Line 729: / Line 729: @@
 Cellulose is a long polysaccharide consisting of β-1,4 linked D-glucose units. Many organisms, including <i>E. coli</i>, are incapable of producing enzymes able to deal with these strong β-linkages. To overcome this obstacle, the <i>Cellumonas fimi</i> developed to express two cellulases; the endo-β-1,4-glucanase (CenA) and the exo-β-1,4-glucanase (Cex) [kilde 7]. The endoglucanase is able to randomly break the amorphous structure of cellulose. This allows for the exoglucanase to break the β-1,4 linkages at every other D-glucose unit, thus releasing disaccharides in the form of cellobiose <span class="reference"><span class="referencetext"><a target="blank" href="https://www.ncbi.nlm.nih.gov/pubmed/9134758/"> Lam TL, Wong RS, Wong WK. Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength. Enzyme and microbial technology. 1997;20(7):482-8.</a></span></span>. Cellulose itself is practically too large to be transported across the cell membrane. This concludes that the breakdown of cellulose into cellobiose, must take place in the extracellular fluid.
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PowerLeaf - a bacterial solar battery

Abstract

About Our Wiki

Achievements

World Situation

Our Solution

Project Design

Light Sensing System

Light Sensing System

Light Sensing System

CO2 fixation

CO2 Fixation

Cellulose production and secretion

Cellulose Production and Secretion

Cellulose Production and Secretion

Breakdown of Cellulose

Optimised nanowires

Optimised Nanowires

Optimised Nanowires

Modelling

Gillespie Algorithm

Toxin/Antitoxin System

Experiments

Demonstration and Results

Parts

Basic Parts

Composite Parts

Notebook

Notebook

SOPs and Protocols

SOPs

Protocols

Safety

Bioethics

Integrated Practices

Education & Public Engagement

Perspectives

To Future iGEM Teams

Team

Attributions

Articles

Websites

Books

Collaboration

Final Words

CO₂ fixation

CO₂ Fixation