Team:Brown-Stanford/PowerCell/Introduction

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== '''Nutrient Secretion''' ==
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== '''Introduction''' ==
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Mars is a hostile, desolate environment.  In order to live there, humans will have to deal with extreme cold, unfiltered solar radiation, low oxygen, and little water{{:Team:Brown-Stanford/Templates/FootnoteNumber|1}}.  Cellular engineering will solve these problems in time, but that raises a new problem--the extra burden of providing these requirements raises the already considerable needs of these microbes{{:Team:Brown-Stanford/Templates/FootnoteNumber|2}}.  It may be possible to feed them from a stored cache of growth nutrients for some time, but these basic requirements will have to be extracted from local resources if a self-sustaining colony is to exist.
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PowerCell is the generator that will power all of the BioTools in a Martian settlement.  
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PowerCell is our solution to this problem; by engineering cyanobacteria to excrete sugar compounds photosynthesized from atmospheric carbon dioxide{{:Team:Brown-Stanford/Templates/FootnoteNumber|3}}, PowerCell will provide other bacterial cultures with a rich carbon source, a basic requirement for producing biomass and other compounds.  In addition, PowerCell able to fix atmospheric N2 and release it in a form accessible to bacteria, providing a basic requirement for protein synthesis and other crucial biological functions{{:Team:Brown-Stanford/Templates/FootnoteNumber|4}}. 
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By producing two of the macromolecules essential to bacterial growth, PowerCell will form a metabolic foundation for the biological systems which will eventually enable a settlement on Mars.  Other microbes producing oxygen, heat, food, light, and other necessities will follow, and in time, a complete biogenic life support system will be put together, all fueled by PowerCell.
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[picture of bio battery]
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[[File:Brown-Stanford PowerCellEnergyFlowDiagram.jpg|700px|center]]
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Like any other tools, biological tools need energy to run and raw materials to work with. The two major nutrient sources needed for biological tools are sugars (carbon sources) and nitrogenous nutrients. In a Martian settlement, PowerCell will harness the energy of the sun and matter from the surrounding atmosphere to provide both these necessities.
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===References===
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{{:Team:Brown-Stanford/Templates/Footnote|1|R. Hanel, B. Conrath, W. Hovis, V. Kunde, P. Lowman, W. Maguire, J. Pearl, J. Pirraglia, C. Prabhakara, B. Schlachman, G. Levin, P. Straat, T. Burke, Investigation of the Martian environment by infrared spectroscopy on Mariner 9, Icarus, Volume 17, Issue 2, October 1972, Pages 423-442, ISSN 0019-1035, DOI: 10.1016/0019-1035(72)90009-7. (http://www.sciencedirect.com/science/article/pii/0019103572900097)}}
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This summer, we tackled only the half involving sugar secretion. We built our system, however, on a platform that can support nitrogen fixation and secretion if someone develops it in the future.  
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{{:Team:Brown-Stanford/Templates/Footnote|2|Weeks, Amy M, and Michelle C Y Chang. 2011. “Constructing de novo biosynthetic pathways for chemical synthesis inside living cells.” Biochemistry 50 (24) (June 21): 5404-5418. doi:10.1021/bi200416g.}}
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[Anabaena picture]
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{{:Team:Brown-Stanford/Templates/Footnote|3|Niederholtmeyer, Henrike, Bernd T Wolfstadter, David F Savage, Pamela A Silver, and Jeffrey C Way. 2010. “Engineering cyanobacteria to synthesize and export hydrophilic products.” Applied and Environmental Microbiology 76 (11) (June): 3462-3466. doi:10.1128/AEM.00202-10.}}
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For our host, we chose a diazotrophic (link to definition) cyanobacterium, Anabaena 7120. Anabaena 7120 has several special characteristics that make it an advantageous host species. First, it can fix its own nitrogen into biologically usable ammonium directly from the atmosphere, and second, it can do this at any time of day. The process of nitrogen fixation is detrimentally affected by the presence of even a small amount of oxygen. For this reason, most diazotrophs separate the processes of photosynthesis and nitrogen fixation temporally – that is, they photosynthesize in the day and fix nitrogen at night. Anabaena 7120 has a different way around this problem. It forms long filaments of cells containing two different cell types, normal (vegetative) cells, and heterocysts. Heterocysts form thick cell walls and house very anaerobic environments; they are where nitrogen fixation takes place. Normal (vegetative) cells carry on with photosynthesis. The two cell types then share nutrients up and down the filament, thus providing each cell with all the nutrients it needs.
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The sugar we chose to secrete is sucrose. The major inspiration for our project was the paper (title, link). This work was done in Dr. Pamela Silver's lab, and describes a glucose/fructose secretion device, achieved in the single-celled bacterium S. elongatus.
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[Silver invA diagram]
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In a nutshell, S. elongatus were forced under salt stress to produce sucrose, which was broken down into glucose and fructose by the glf enzyme, then transported out of the cell by the invA transporter.
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At the Fifth International Conference on Synthetic Biology, SB5.0, (link to outreach page) we spoke with a member of Dr. Silver's lab, Danny Ducat. He advised us that one of the problems with the glucose/fructose secretion system is its low yield. He suspected that because glucose and fructose are directly metabolizable by the cell, much of these sugars are consumed by the cell before they ever have a chance to be secreted. For this reason, it may be better to directly secrete sucrose, which is not metabolizable by the cell, and worry about breaking it down later. This is what we did.
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Below is a diagram of our sucrose secretion device. We placed the sucrose symporter gene, cscB, behind a promoter present in the Photo-system I of Anabaena 7120. This was done to confine sucrose secretion to only vegetative cells, as heterocysts are not producing sucrose through photosynthesis.
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[Ana-cscB diagram]
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We have also created several other constructs containing GFP for debugging purposes. We used a modified version of GFP, GFPmut3B because normal florescence markers are easily lost among background chlorophyll pigments in cyanobacteria.
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[Ana-GFP diag]
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[Ana-cscB-GFP diag]
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One major challenge was transforming our constructs into Anabaena 7120. Anabeana has several restriction enzymes, AvaI, II, and III, which prove problematic for routine transformations into the species. The method required for such a transformation was developed by Elhai and Wolk (cite paper). It requires a conjugal mating of the target cyanobacterium with two strains of E. coli, containing the plasmid of interest and a conjugal transfer plasmid, respectively.
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=== '''Utilizing sucrose''' ===
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{{:Team:Brown-Stanford/Templates/Footnote|4|Chaurasia, Akhilesh Kumar, and Shree Kumar Apte. 2011. “Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential.” Applied and Environmental Microbiology 77 (2) (January): 395-399. doi:10.1128/AEM.01714-10.}}
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Revision as of 17:03, 27 September 2011

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Introduction

Mars is a hostile, desolate environment. In order to live there, humans will have to deal with extreme cold, unfiltered solar radiation, low oxygen, and little water1. Cellular engineering will solve these problems in time, but that raises a new problem--the extra burden of providing these requirements raises the already considerable needs of these microbes2. It may be possible to feed them from a stored cache of growth nutrients for some time, but these basic requirements will have to be extracted from local resources if a self-sustaining colony is to exist.

PowerCell is our solution to this problem; by engineering cyanobacteria to excrete sugar compounds photosynthesized from atmospheric carbon dioxide3, PowerCell will provide other bacterial cultures with a rich carbon source, a basic requirement for producing biomass and other compounds. In addition, PowerCell able to fix atmospheric N2 and release it in a form accessible to bacteria, providing a basic requirement for protein synthesis and other crucial biological functions4.

By producing two of the macromolecules essential to bacterial growth, PowerCell will form a metabolic foundation for the biological systems which will eventually enable a settlement on Mars. Other microbes producing oxygen, heat, food, light, and other necessities will follow, and in time, a complete biogenic life support system will be put together, all fueled by PowerCell.

Brown-Stanford PowerCellEnergyFlowDiagram.jpg

References

1 R. Hanel, B. Conrath, W. Hovis, V. Kunde, P. Lowman, W. Maguire, J. Pearl, J. Pirraglia, C. Prabhakara, B. Schlachman, G. Levin, P. Straat, T. Burke, Investigation of the Martian environment by infrared spectroscopy on Mariner 9, Icarus, Volume 17, Issue 2, October 1972, Pages 423-442, ISSN 0019-1035, DOI: 10.1016/0019-1035(72)90009-7. (http://www.sciencedirect.com/science/article/pii/0019103572900097)

2 Weeks, Amy M, and Michelle C Y Chang. 2011. “Constructing de novo biosynthetic pathways for chemical synthesis inside living cells.” Biochemistry 50 (24) (June 21): 5404-5418. doi:10.1021/bi200416g.

3 Niederholtmeyer, Henrike, Bernd T Wolfstadter, David F Savage, Pamela A Silver, and Jeffrey C Way. 2010. “Engineering cyanobacteria to synthesize and export hydrophilic products.” Applied and Environmental Microbiology 76 (11) (June): 3462-3466. doi:10.1128/AEM.00202-10.

4 Chaurasia, Akhilesh Kumar, and Shree Kumar Apte. 2011. “Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential.” Applied and Environmental Microbiology 77 (2) (January): 395-399. doi:10.1128/AEM.01714-10.

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