Team:Hong Kong-CUHK/Project

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<tr><td > <h3> Project Description </h3></td>
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<td ></td >
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<td > <h3> Content</h3></td>
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</tr>
</tr>
<tr>
<tr>
<td width="45%"  valign="top">  
<td width="45%"  valign="top">  
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<p>Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) </p>
+
<b style='mso-bidi-font-weight:normal'><span lang=EN-US>Methanator</span></b></span></p>
-
<br>
+
 
-
<h3>References </h3>
+
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><b
-
<p>
+
style='mso-bidi-font-weight:normal'><i style='mso-bidi-font-style:normal'><span
-
iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>  
+
lang=EN-US>Project Description</span></i></b></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>Carbon dioxide (CO<sub>2</sub>) 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 <span class=SpellE>lifes</span> (<span class=SpellE>Baldgcchi</span>
 +
<i style='mso-bidi-font-style:normal'>et al.</i>, 1996). This year we would
 +
like to utilize and recharge these abundantly available CO<sub>2</sub> by
 +
converting to methane (CH<sub>4</sub>), an important carbon source for fuel and
 +
bio-degradable plastic production. While there are naturally existing
 +
methane-generating microorganisms, the <span class=SpellE>convertion</span>
 +
involves multi-step metabolic reactions, not to mention that they can only
 +
survive in anaerobic environment thus <span class=SpellE>diffcult</span> to
 +
manipulate.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>A recent research showed that a mutated form of <span class=SpellE>nitrogenase</span>
 +
from <span class=SpellE><i style='mso-bidi-font-style:normal'>Azotobacter</i></span><i
 +
style='mso-bidi-font-style:normal'> <span class=SpellE>vinelandii</span></i>, a
 +
nitrogen-fixing bacteria found in soil, has carbon fixation ability (<span
 +
class=SpellE>Seefeldt</span> <i style='mso-bidi-font-style:normal'>et al.</i>,
 +
2013). Yang <i style='mso-bidi-font-style:normal'>et al. </i>demonstrated that
 +
by introducing 70Ala and 195Gln mutations on <span class=SpellE>nitrogenase</span>
 +
alpha subunit, the <span class=SpellE><span class=GramE>nitrogenase</span></span>
 +
enzyme complex reduced CO<sub>2</sub> and CO<sub>3</sub><sup>-</sup> to CH<sub>4</sub>
 +
instead of converting nitrogen to ammonia (Yang <i style='mso-bidi-font-style:
 +
normal'>et al.</i>, 2012). This system provided <span class=GramE>an</span>
 +
one-step reaction to convert CO<sub>2</sub> into CH<sub>4</sub> and other
 +
carbon compounds directly. However, since a large electron flux, thus energy,
 +
was wasted in producing molecular hydrogen (H<sub>2</sub>) from proton during
 +
the reaction, we utilized a soluble <span class=SpellE>hydrogenase</span>
 +
complex from <span class=SpellE><i style='mso-bidi-font-style:normal'>Aquifex</i></span><i
 +
style='mso-bidi-font-style:normal'> <span class=SpellE>aeolicus</span></i> to
 +
recycle H<sub>2</sub> to proton. To further enhance the efficiency of carbon
 +
fixation process, we physically linked both <span class=SpellE>nitrogenase</span>
 +
and <span class=SpellE>hydrogenase</span> complexes with SH3 and PDZ
 +
ligand-domain pairs to accelerate the H<sub>2</sub> recycling.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>To allow regulation of expression of these enzymes in <i
 +
style='mso-bidi-font-style:normal'>A. <span class=SpellE>vinelandii</span></i>,
 +
we plan to build a T7 protein expression system in <span class=GramE>this <span
 +
class=SpellE>diazotrophic</span> bacteria</span>. Given that this organism
 +
produces more <span class=SpellE>nitrogenase</span> complex in the absence of
 +
ammonia (<span style='color:#222222;background:white;mso-highlight:white'>Hamilton
 +
<i style='mso-bidi-font-style:normal'>et al</i>., 2011</span>)<span
 +
class=GramE>,</span> we utilized its strong nitrogen-<span class=SpellE>derepressible</span>
 +
<span class=SpellE>nifH</span> promoter (Trinity <i style='mso-bidi-font-style:
 +
normal'>et al.</i>, 2011) to control T7 RNA polymerase expression. <span
 +
class=SpellE><span class=GramE>nifH</span></span> promoter appeared to be
 +
stronger than its counterpart, lacUV5 promoter, in <i style='mso-bidi-font-style:
 +
normal'>E. coli </i>(<span class=SpellE>Curatti</span>, <i style='mso-bidi-font-style:
 +
normal'>et al.</i>, 2005) (IGEM, 2011). Our system may provide an alternative
 +
platform for protein expression, as it is compatible to existing T7
 +
promoter-driven constructs. Expression of enzymes along a particular metabolic
 +
pathway becomes possible as stable genome integration of DNA up to several <span
 +
class=SpellE>MBps</span> could be done in <i style='mso-bidi-font-style:normal'>A.
 +
<span class=SpellE>vinelandii</span> </i>(Catherine,<i style='mso-bidi-font-style:
 +
normal'> et al.</i>, 1989). Moreover, we only need ammonia to repress the
 +
expression instead of using expensive IPTG in the counterpart, thus lowering
 +
the expense.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><span style='mso-spacerun:yes'>&nbsp;</span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>To sum up, two goals will be achieved: to create a carbon fixation
 +
system using a combination of bacterial <span class=SpellE>nitrogenase</span>
 +
and <span class=SpellE>hydrogenase</span> enzyme complexes to convert CO2 to
 +
CH4, and to develop a novel nitrogen-regulated T7 protein expression system. </span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><b
 +
style='mso-bidi-font-weight:normal'><i style='mso-bidi-font-style:normal'><span
 +
lang=EN-US>References</span></i></b></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>1.<span style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp; </span><span
 +
class=SpellE>Seefeldt</span>, L. C., Yang, Z. Y., Duval, S., &amp; Dean, D. R.
 +
2013. <span class=SpellE><span class=GramE>Nitrogenase</span></span><span
 +
class=GramE> reduction of carbon-containing compounds.</span> <span
 +
class=SpellE><i style='mso-bidi-font-style:normal'>Biochimica</i></span><i
 +
style='mso-bidi-font-style:normal'> <span class=GramE>et</span> <span
 +
class=SpellE>Biophysica</span> <span class=SpellE>Acta</span>
 +
(BBA)-Bioenergetics</i>, <i style='mso-bidi-font-style:normal'>1827</i>(8),
 +
1102-1111.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>2.<span style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span>BALDOCCHI, D., VALENTINI, R., RUNNING, S., OECHEL, W., &amp; DAHLMAN, R.
 +
1996. <span class=GramE>Strategies for measuring and <span class=SpellE>modelling</span>
 +
carbon dioxide and water <span class=SpellE>vapour</span> fluxes over
 +
terrestrial ecosystems.</span> <span class=GramE><i style='mso-bidi-font-style:
 +
normal'>Global change biology</i>, <i style='mso-bidi-font-style:normal'>2</i>(3),
 +
159-168.</span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>3. Yang, Z. Y., Moure, V. R., Dean, D. R., &amp; <span class=SpellE>Seefeldt</span>,
 +
L. C. (2012). Carbon dioxide reduction to methane and coupling with acetylene
 +
to form propylene catalyzed by remodeled <span class=SpellE>nitrogenase</span>.
 +
<i style='mso-bidi-font-style:normal'>Proceedings of the National Academy of
 +
Sciences</i>, <i style='mso-bidi-font-style:normal'>109</i>(48), 19644-19648.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>4.<span style='color:#222222;background:white;mso-highlight:white'>Hamilton,
 +
T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos Santos, P. C., Setubal, J. C.,
 +
... &amp; Peters, J. W. (2011). <span class=GramE>Transcriptional profiling of
 +
nitrogen fixation in <span class=SpellE>Azotobacter</span> <span class=SpellE>vinelandii</span>.</span>
 +
<span class=GramE><i style='mso-bidi-font-style:normal'>Journal of bacteriology</i>,
 +
<i style='mso-bidi-font-style:normal'>193</i>(17), 4477-4486.</span></span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>5. Trinity L. H., Marcus L., Ray <span class=SpellE>D.,Eric</span> <span
 +
class=SpellE>S.B.,Patricia</span> C.D.S., <span class=SpellE>Jo&atilde;o</span> C. S.,
 +
Donald A. B., Dennis R. D. and John W. P., (2011). Transcriptional Profiling of
 +
Nitrogen Fixation in <span class=SpellE>Azotobacter</span> <span class=SpellE>vinelandii</span>.
 +
<i style='mso-bidi-font-style:normal'>Journal of Bacteriology</i>, 193(17):
 +
4477–4486.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><span style='mso-spacerun:yes'>&nbsp;</span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
class=GramE><span lang=EN-US>6.Curatti</span></span><span lang=EN-US>, L.,
 +
Brown, C. S., <span class=SpellE>Ludden</span>, P. W., &amp; Rubio, L. M.
 +
(2005). Genes required for rapid expression of <span class=SpellE>nitrogenase</span>
 +
activity in <span class=SpellE>Azotobacter</span> <span class=SpellE>vinelandii.<i
 +
style='mso-bidi-font-style:normal'>Proceedings</i></span><i style='mso-bidi-font-style:
 +
normal'> of the National Academy of Sciences of the United States of America</i>,
 +
<i style='mso-bidi-font-style:normal'>102</i>(18), 6291-6296.</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><span style='mso-spacerun:yes'>&nbsp;</span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>7. http://parts.igem.org/Part:BBa_K568003</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><span style='mso-spacerun:yes'>&nbsp;</span></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US>8. Catherine S. R., J. J. Pasternak, Bernard R. G., (1989). <span
 +
class=GramE>Integration of exogenous DNA into the genome of <span class=SpellE>Azotobacter</span>
 +
<span class=SpellE>vinelandii</span>.</span> Archives of <span class=GramE>Microbiology
 +
,152</span>(5), 437-440</span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-align:justify;text-justify:inter-ideograph'><span
 +
lang=EN-US><o:p>&nbsp;</o:p></span></p>
</td>
</td>

Revision as of 12:59, 14 August 2014



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Methanator

Project Description

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 to methane (CH4), an important carbon source for fuel and bio-degradable plastic production. While there are naturally existing methane-generating microorganisms, the convertion involves multi-step metabolic reactions, not to mention that they can only survive in anaerobic environment thus diffcult to manipulate.

 

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 CO3- to CH4 instead of converting nitrogen to ammonia (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, thus energy, was wasted in producing molecular hydrogen (H2) from proton during the reaction, we utilized a soluble hydrogenase complex from Aquifex aeolicus to recycle H2 to proton. 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.

 

To allow regulation of expression of these enzymes in A. vinelandii, we plan to build a T7 protein expression system in this diazotrophic bacteria. Given that this organism produces more nitrogenase complex in the absence of ammonia (Hamilton et al., 2011), we utilized its strong nitrogen-derepressible nifH promoter (Trinity et al., 2011) to control T7 RNA polymerase expression. nifH promoter appeared to be stronger than its counterpart, lacUV5 promoter, in E. coli (Curatti, et al., 2005) (IGEM, 2011). Our system may provide an alternative platform for protein expression, as it is compatible to existing T7 promoter-driven constructs. Expression of enzymes along a particular metabolic pathway becomes possible as stable genome integration of DNA up to several MBps could be done in A. vinelandii (Catherine, et al., 1989). Moreover, we only need ammonia to repress the expression instead of using expensive IPTG in the counterpart, thus lowering the expense.

 

To sum up, two goals will be achieved: to create a carbon fixation system using a combination of bacterial nitrogenase and hydrogenase enzyme complexes to convert CO2 to CH4, and to develop a novel nitrogen-regulated T7 protein expression system.

 

References

1.    Seefeldt, L. C., Yang, Z. Y., Duval, S., & Dean, D. R. 2013. Nitrogenase reduction of carbon-containing compounds. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1827(8), 1102-1111.

2.     BALDOCCHI, D., VALENTINI, R., RUNNING, S., OECHEL, W., & DAHLMAN, R. 1996. Strategies for measuring and modelling carbon dioxide and water vapour fluxes over terrestrial ecosystems. Global change biology, 2(3), 159-168.

 

3. Yang, Z. Y., Moure, V. R., Dean, D. R., & Seefeldt, L. C. (2012). Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase. Proceedings of the National Academy of Sciences, 109(48), 19644-19648.

 

4.Hamilton, T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos Santos, P. C., Setubal, J. C., ... & Peters, J. W. (2011). Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii. Journal of bacteriology, 193(17), 4477-4486.

 

5. Trinity L. H., Marcus L., Ray D.,Eric S.B.,Patricia C.D.S., João C. S., Donald A. B., Dennis R. D. and John W. P., (2011). Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii. Journal of Bacteriology, 193(17): 4477–4486.

 

6.Curatti, L., Brown, C. S., Ludden, P. W., & Rubio, L. M. (2005). Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.Proceedings of the National Academy of Sciences of the United States of America, 102(18), 6291-6296.

 

7. http://parts.igem.org/Part:BBa_K568003

 

8. Catherine S. R., J. J. Pasternak, Bernard R. G., (1989). Integration of exogenous DNA into the genome of Azotobacter vinelandii. Archives of Microbiology ,152(5), 437-440

 

 

 

 

You can use these subtopics to further explain your project

  1. Overall project summary
  2. Project Details
  3. Materials and Methods
  4. The Experiments
  5. Results
  6. Data analysis
  7. Conclusions

It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.

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