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| | == Project Details== | | == Project Details== |
| - | The project will focus on the creation of an '''efficient and cost effective''' biological battery, the BioBattery. The BioBattery will be created with the use of cyanobacterium, ''Spirulina maxima'', a photosynthetic blue-green algae, which has been shown to produce an electrical current. ''S. maxima'' has been shown to generate up to 800 mV of electricity per cm^2, and although this does essentially provide enough energy to charge a battery, it may simply not be enough to sustain the battery once it is being used. Therefore, we must optimize its electrical output and create the BioBattery.
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| - | To optimize the BioBattery, we will be utilizing the microcompartment found and characterized from ''Aquifex aeolicus''. This protein will be optimized for the targeting of desired materials into the compartment. The generation of such a microcompartment would act to revolutionize the synthetic biology approach, allowing for easier cloning strategies, bringing enzymes from various organisms which might not normally work in conjunction into close proximity, minimizing side reactions and allowing for the formation of efficient novel metabolic pathways. There are sub-projects which will be completed on the way to the final goal of the production of the BioBattery which are outlined as:
| + | Many of the metabolic processes in eukaryotic cells are localized to various subcellular compartments. For example, transcription of mRNA from DNA is localized in the nucleus; post-translational modification and folding of proteins occurs in the endoplasmic reticulum (Voet et al., 2004). [[Image:YFP_CFP_Lumazine.png|right|275px]] This essentially segregates and regulates these processes. However, bacteria lack distinct organelles, and as such, many metabolic processes intermingle. Engineering an artificial organelle, capable of containing metabolic proteins, in bacteria would thus represent a fundamental advance in biotechnology. Towards this end, the University of Lethbridge iGEM team is working towards producing microcompartments from the lumazine synthase gene, which forms 60 subunit icosahedral capsids (Seebeck et al., 2006). [[Image:YFP_CFP_fret.png|left|275px]]By generating a highly negative interior to the microcompartment, we hope to be able to target fluorescent proteins tagged with positively charged termini to the interior of the microcompartment (Figure 1). Utilizing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins, we will demonstrate the co-localization of these two proteins into the microcompartment (Figure 2). For the future application of this technology we are working towards the targeting of proteins from the photosynthetic pathway into the microcompartment to optimize a biological photosynthetic fuel cell. The segregation of proteins and metabolites increases the efficiency of cellular processes and following this theme, we are working towards creating uniform nanoparticles through the segregation of Mms6 protein within Escherichia coli (Figure 3) |
| | + | [[Image:Nanoparticles.png|right|350px]] |
| | + | (Prozorov et al., 2007). |
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| - | 1) The compartmentalization of cellular photosystems:
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| - | [[Image:YFP_CFP_Lumazine.png|right|275px]]
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| - | The lumazine synthase protein of ''A. aeolicus'' (ribH gene) has been shown to produce 60 subunit icosahedral capsids. Towards this end, Seebeck and colleagues were able to engineer an electronegative surface in the pore of the assembled capsid [Seebeck et al. (2006) J. Am. Eng. Soc.]. By attaching a positively charged tail onto the terminus of a fluorescent protein, the protein was targeted into the capsid, and its fluorescence was observed. We will attach arginine tags (10X Arg tags) to the termini of proteins to be targeted, as well as a synthesized biobrick encompassing the ribH gene. We intend to demonstrate co-localization of two fluorescent proteins (cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)) to the interior of the capsid using fluorescent resonance energy transfer (FRET). This will be used as a proof of principle system prior to introduction of proteins to increase the efficiency of the BioBattery.
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| - | [[Image:YFP_CFP_fret.png|left|275px]]
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| - | 2) Novel electron facilitators
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| - | Upon successful targeting, it is feasible that the incorporation of a novel electron shuttling protein into the engineered microcompartment would increase the current generated by facilitating electron movement to the electrodes of the microbial fuel cell. Indeed, many MFCs use mediators to increase their efficiency; however, these substances tend to be expensive. Towards this end, we have identified the gene encoding for '''naphthalene dioxygenase, ndoB'''. This protein is involved in the formation of humic acid, a common electron mediator, in soil bacteria such as ''Pseudomonas aeruginosa''.
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| - | 3) Production of nanoparticles
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| - | Many applications which require the use of nanoparticles, such as medical and diagnostic sciences, require that the particles adopt uniform structures and sizes.[[Image:Nanoparticles.png|right|350px]] Current methods used to produce these nanoparticles are cost intensive and require extreme conditions and harsh organic solvents [Amemiya et al. (2007) Biomaterials]. However, bacterial strains have been found that produce these nanoparticles with '''specific sizes and distinct morphologies'''. The proteins involved in the process the nanoparticle production are being characterized to create a better understanding of the process. A protein of particular interest to us is '''mms6''', produced by ''Magnetopirillum magneticum'', has been found to be key in '''controlling the morphology of the particles'''. This protein shall be introduced into E. coli with the intention introducing a novel method for the mass production uniform nanoparticles which is '''efficient and cost effective'''.
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| - | ===Refernces===
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| - | Prozorov, T., Mallapragada, S. K., Narasimhan, B., Wang, L., Palo, P.,
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| - | Nilsen-Hamilton, M., Williams, T. J., Bazylinski, D. A., Prozorov, R., and
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| - | Canfield, P. C. (2007). Protein-mediated synthesis of uniform
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| - | superparamagnetic magnetite nanocrystals. Adv. Funct. Mater. Advanced
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| - | Functional Materials. 17, 951-957
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| - | Seebeck, F. P., Woycechowsky, K. J., Zhuang, W., Rabe, J. P., and Hilvert,
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| - | D., (2006). A simple tagging system for protein encapulation. J. Am. Chem.
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| - | Soc. 128, 4516-4517
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| - | Tsujimura, S., Wadano, A., Kano, K., and Ikeda, T., (2001). Photosynthetic
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| - | bioelectrochemical cell utilizing cyanobacteria and water-generating
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| - | oxidase. Enzyme and Microbial Technology. 29, 225-231
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Many of the metabolic processes in eukaryotic cells are localized to various subcellular compartments. For example, transcription of mRNA from DNA is localized in the nucleus; post-translational modification and folding of proteins occurs in the endoplasmic reticulum (Voet et al., 2004).
This essentially segregates and regulates these processes. However, bacteria lack distinct organelles, and as such, many metabolic processes intermingle. Engineering an artificial organelle, capable of containing metabolic proteins, in bacteria would thus represent a fundamental advance in biotechnology. Towards this end, the University of Lethbridge iGEM team is working towards producing microcompartments from the lumazine synthase gene, which forms 60 subunit icosahedral capsids (Seebeck et al., 2006).
By generating a highly negative interior to the microcompartment, we hope to be able to target fluorescent proteins tagged with positively charged termini to the interior of the microcompartment (Figure 1). Utilizing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins, we will demonstrate the co-localization of these two proteins into the microcompartment (Figure 2). For the future application of this technology we are working towards the targeting of proteins from the photosynthetic pathway into the microcompartment to optimize a biological photosynthetic fuel cell. The segregation of proteins and metabolites increases the efficiency of cellular processes and following this theme, we are working towards creating uniform nanoparticles through the segregation of Mms6 protein within Escherichia coli (Figure 3)
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