Team:Hong Kong-CUHK/projectA-background.html

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<h2>Background</h2>
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<h2>Why Azotobacter Vinelandii?</h2>
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<p>Although Azotobacter Vinelandii is not commonly used as a model of gram-negative bacteria, there are many features that make us introduce the chassis to iGEM. We think that it is very suitable for genetic engineering based on the features below:</p>
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    Carbon dioxide (CO2) is notorious for its major contribution to global warming, where one of the impacts brought to the ecosystem is its excessive solvation into the ocean in carbonate form, threatening marine lifes (Baldgcchi et al., 1996). This year we would like to utilize and recharge these abundantly available CO2 by converting it to methane (CH4), an important carbon source for fuels and bio-degradable plastic production. While there are naturally existing methane-generating microorganisms, the convertion process involves multi-step metabolic reactions, not to mention that the mircoorganisms can only survive in anaerobic environment. Therefore, the difficulty of manipulating this convertion process remains high.
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<li>Anaerobic intracellular environment in aerobic extracellular environment which could use to express oxygen sensitive protein</li>
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<li>Most parts in registry optimized for E. coli may also function</li>
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<li>Can use stable genome integration which could transform a larger size of gene at once</li>
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<li>Antibiotic such as ampicillin and kanamycin can be used</li>
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<p>For introducing the stable genome integration, we constructs several biobricks. Moreover, as many promoter of registry cannot be used in Azotobacter Vinelandii, we introduce a novel T7 dependent system using nifH promote. Features about the protein expression system:<p>
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    A recent research showed that a mutated form of nitrogenase from Azotobacter vinelandii, a nitrogen-fixing bacteria found in soil, has carbon fixation ability (Seefeldt et al., 2013). Yang et al. demonstrated that by introducing 70Ala and 195Gln mutations on nitrogenase alpha subunit, the nitrogenase enzyme complex reduced CO2 and CO32- to CH4 instead of converting N2 to NH3 (Yang et al., 2012). This system provided an one-step reaction to convert CO2 into CH4 and other carbon compounds directly. However, since a large electron flux, and thus energy, was wasted in producing molecular hydrogen (H2) from protons during the reaction, we utilized a soluble hydrogenase complex from Aquifex aeolicus to recycle H2 to protons. To further enhance the efficiency of carbon fixation process, we physically linked both nitrogenase and hydrogenase complexes with SH3 and PDZ ligand-domain pairs to accelerate the H2 recycling.
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<li>Nitrogen inducible—repressed by ammonia and other nitrogen source</li>
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<li>Predicted to express better than the original T7 expression system</li>
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<li>Make T7 promoter usable in Azotobacter Vinelandii, and hence, make a lot more biobricks could be used in Azotobacter Vinelandii.</li>
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Latest revision as of 04:13, 27 November 2014

Why Azotobacter Vinelandii?

Although Azotobacter Vinelandii is not commonly used as a model of gram-negative bacteria, there are many features that make us introduce the chassis to iGEM. We think that it is very suitable for genetic engineering based on the features below:

    1. Anaerobic intracellular environment in aerobic extracellular environment which could use to express oxygen sensitive protein
    2. Most parts in registry optimized for E. coli may also function
    3. Can use stable genome integration which could transform a larger size of gene at once
    4. Antibiotic such as ampicillin and kanamycin can be used


For introducing the stable genome integration, we constructs several biobricks. Moreover, as many promoter of registry cannot be used in Azotobacter Vinelandii, we introduce a novel T7 dependent system using nifH promote. Features about the protein expression system:<p>

    1. Nitrogen inducible—repressed by ammonia and other nitrogen source
    2. Predicted to express better than the original T7 expression system
    3. Make T7 promoter usable in Azotobacter Vinelandii, and hence, make a lot more biobricks could be used in Azotobacter Vinelandii.

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