Team:Bielefeld-CeBiTec/Results/Outlook

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<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol">Here</a> you will find information about the execution of our experiments.
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<h6>Isobutanol pathway</h6>
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Building on the results shown in the section <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/Pathway">Isobutanol pathway</a> further analysis could follow. We could show via SDS Page that potentially all proteins are expressed. Furthermore we could demonstrate that bacteria carrying our constructs produce isobutanol.
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  <h4>Intermediates</h4>
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It turns out the the cultures carrying the construct <a href="http://parts.igem.org/Part:BBa_K1465307" target="_blank">BBa_K1465307</a> (adhA containing) produced less isobutanol than cultures carrying the construct <a href="http://parts.igem.org/Part:BBa_K1465306" target="_blank">BBa_K1465306</a>. Regarding this we conclude that the used AdhA did not improve isobutanol production contrary to the literature. To detect possible causes for this phenomena, it would be helpful to analyze the intermediates of the used pathway. (<a href="#Atsumi2008">Atsumi et al., 2008</a>) There might be a difference between the amounts of the particular intermediates which could explain the different production rates as well as help detecting metabolic bottlenecks.
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  <h4>Conditions</h4>
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Furthermore an increase of the production rate would be an aim of further experiments. Therefore optimized conditions have to be identified. Maybe a temperature between 30°C and 37°C could be tryed out.
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  <h4>Pathway</h4>
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A further approach could be the implementation of other genes for the 2-keto-acid pathway, such as the <i>kivD</i> from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis"><i>Lactococcus lactis</i></a>  in our isobutanol production pathway.
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  <h4>Separation</h4>
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For analyzing our isobutanol production we took samples directly of the cultures and analyzed the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#GC-MS" target="_blank">supernatant</a>. The next step would be the separation of isobutanol and the medium respectively the bacteria during the cultivation. This could be done by the implementation of a dialysis system. The iGEM team NCTU Formosa presented a <a href="https://2012.igem.org/Team:NCTU_Formosa/Project-sub2#sub2-4" target="_blank">possible system</a>.
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<h6>Alcohol dehydrogenase</h6>
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As described in the section about the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/AdhA">alcohol dehydrogenase</a> we could show the successful overexpression of the protein AdhA from <i>L. lactis</i> in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli"><i>E. coli</i></a>.
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  <h4>Anaerobic cultivation</h4>
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Further analysis could be an anaerobic cultivation for a complementation analysis of the strain <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/StrainsAndConstructs#DeltaAdh">&Delta;adhE748::kan</a>. <a href="#Trinh2011">Trinh et al., 2011</a> showed that <i>E. coli</i> cannot grow after the loss of its alcohol dehydrogenase. Complemented with our plasmid (<a href="BBa_K1465305" target="_blank">BBa_K1465305</a>) the mutant should grow. This experiment setup would be a possible in vivo functionality test of our AdhA.
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              <p class="buttoncenter"><font color="#FFFFFF">Overview</font></p>
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  <h6>References</h6>
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  Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: <a href="http://www.nature.com/nature/journal/v451/n7174/full/nature06450.html" target="_blank">Nature 451</a>, 86–89.
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  Trinh CT, Li J, Blanch HW, Clark DS., 2011. Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol. In: <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147371/" target="_blank">Appl Environ Microbiol.</a>, 77(14): 4894-904
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Latest revision as of 01:20, 18 October 2014


Module III - Isobutanol production

Isobutanol pathway
Building on the results shown in the section Isobutanol pathway further analysis could follow. We could show via SDS Page that potentially all proteins are expressed. Furthermore we could demonstrate that bacteria carrying our constructs produce isobutanol.

Intermediates

It turns out the the cultures carrying the construct BBa_K1465307 (adhA containing) produced less isobutanol than cultures carrying the construct BBa_K1465306. Regarding this we conclude that the used AdhA did not improve isobutanol production contrary to the literature. To detect possible causes for this phenomena, it would be helpful to analyze the intermediates of the used pathway. (Atsumi et al., 2008) There might be a difference between the amounts of the particular intermediates which could explain the different production rates as well as help detecting metabolic bottlenecks.

Conditions

Furthermore an increase of the production rate would be an aim of further experiments. Therefore optimized conditions have to be identified. Maybe a temperature between 30°C and 37°C could be tryed out.

Pathway

A further approach could be the implementation of other genes for the 2-keto-acid pathway, such as the kivD from Lactococcus lactis in our isobutanol production pathway.

Separation

For analyzing our isobutanol production we took samples directly of the cultures and analyzed the supernatant. The next step would be the separation of isobutanol and the medium respectively the bacteria during the cultivation. This could be done by the implementation of a dialysis system. The iGEM team NCTU Formosa presented a possible system.

Alcohol dehydrogenase

As described in the section about the alcohol dehydrogenase we could show the successful overexpression of the protein AdhA from L. lactis in E. coli.

Anaerobic cultivation

Further analysis could be an anaerobic cultivation for a complementation analysis of the strain ΔadhE748::kan. Trinh et al., 2011 showed that E. coli cannot grow after the loss of its alcohol dehydrogenase. Complemented with our plasmid (BBa_K1465305) the mutant should grow. This experiment setup would be a possible in vivo functionality test of our AdhA.


References
  • Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.
  • Trinh CT, Li J, Blanch HW, Clark DS., 2011. Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol. In: Appl Environ Microbiol., 77(14): 4894-904

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