Team:Uppsala-Sweden/Ethanol

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The Pathway

The idea to produce ethanol with the use of cyanobacteria was originally proposed and already accomplished by Ming-De Deng and John R. Coleman. [1] We from the Uppsala iGEM Team decided to try to improve the production of ethanol by interfering with the metabolic pathways of Synechocystis sp. PCC 6803 by an Antisense RNA and a protein-mediated approach. Hence we build a construct that encodes for ethanol production in our host organism. But first of all the some theory about the molecular mechanism.

Light energy is used in the Calvin cycle to transform water and carbon dioxide into organic compounds, among those is Ribulose-1,5-bisphosphate, which immediately splits into two 3-Phosphoglycerate molecules. [2] These two molecules can now be converted to 2-Phosphoglycerate. Enolase and pyruvatekinase finally catalyze then the reaction over phosphoenolpyruvate to pyruvate. [3] This pyruvate molecule is now substrate for the ethanol production.

blabal mechanism

References

[1] Ethanol synthesis by genetic engineering in cyanobacteria. Deng MD, Coleman JR. Appl Environ Microbiol. 1999 Feb;65(2):523-8

[2] Jeremy M. Berg, John L. Tymoczko, L. Stryer Biochemistry 5th Edition; Ch.21.1 p826-838<i> 653

[3] Jeremy M. Berg, John L. Tymoczko, L. Stryer <i>Biochemistry 5th Edition; Ch.16.1 p653<i>

[4] MetaCyc Reaction: 4.1.1.1

[5] MetaCyc Reaction: 1.1.1.1

@@ Line 1: / Line 1: @@
-{{Uppsala-Sweden Template}}
+==The Pathway==
-==The Ethanol Project==
+The idea to produce ethanol with the use of cyanobacteria was originally proposed and already accomplished by Ming-De Deng and John R. Coleman. [[#ref1|<nowiki>[1]</nowiki>]] We from the Uppsala iGEM Team decided to try to improve the production of ethanol by interfering with the metabolic pathways of <i>Synechocystis sp. PCC 6803</i> by an [https://2009.igem.org/Team:Uppsala-Sweden/Ethanol Antisense RNA] and a [https://2009.igem.org/Team:Uppsala-Sweden/rna protein-mediated] approach. Hence we build a construct that encodes for ethanol production in our host organism. But first of all the some theory about the molecular mechanism.
-Our goal is to introduce ethanol producing capability into a cyanobacteria using the biobrick system.
-==How to ==
-Cyanobacteria have the capability to harvest the energy from the sun and convert it into other forms of energy. The natural way for these organisms is to store it as sugars or other carbohydrates in a way similar to plants.
-By introducing a casette of genes for ethanol production, we could get a cyanobacteria to start producing ethanol. However the yield from this might be very low since the bacteria have already evolved good pathways in the metabolism to take care of the energy.
-The source of carbon for the ethanol is pyruvate which is also used in the Krebs cycle. To get a higher yield the pyruvate pool must be large enough for the ethanol anabolism. By blocking the pyruvate dehydrogenase the usage of pyruvate in the Krebs cycle can be reduced and the pyruvate used for ethanol production instead.
-==The Constructs==
-We designed multiple constructs as we ran into some unexpected restriction site problem during the assembling process. The <i>Z. mobilis</i> strain which was source of the pdc and adh, most probably had a mutation inside the adh2 gene, which lead to a novel EcoRI restriction site. Thus we decided to build a back up construct using the adh2 from <i>S. cerevisiae</i>.
-We build as well versions for testing purposes in <i>E. Coli</i>.
-<h3><partinfo>BBa_K273056</partinfo> : The Construct for Testing Purposes in <i>E. Coli</i> with adh2 from <i>Z. mobilis</i></h3>
-<center>[[Image:ethanol1_ecoli.png]]</center>
-<h3><partinfo>BBa_K273057</partinfo> : The Final Construct for <i>Synechocystis sp PCC6803</i> with adh2 from <i>Z. mobilis</i></h3>
-<center>[[Image:ethanol1.png]]</center>
-<h3><partinfo>BBa_K273015</partinfo> : The Construct for Testing Purposes in <i>E. Coli</i> with adh2 from <i>S. cerevisiae</i></h3>
-<center>[[Image:ethanol2_ecoli.png]]</center>
-<h3><partinfo>BBa_K273016</partinfo> : The Final Construct for <i>Synechocystis sp PCC6803</i> with adh2 from <i>S. cerevisiae</i></h3>
-<center>[[Image:ethanol2.png]]</center>
-==The Pathway==
 <center>
 [[Image:ethanolpath_horizontal.png]]
 </center>
-Here some theory...
+Light energy is used in the Calvin cycle to transform water and carbon dioxide into organic compounds, among those is Ribulose-1,5-bisphosphate, which immediately splits into two 3-Phosphoglycerate molecules. [[#ref2|<nowiki>[2]</nowiki>]] These two molecules can now be converted to 2-Phosphoglycerate. Enolase and pyruvatekinase finally catalyze then the reaction over phosphoenolpyruvate to pyruvate. [[#ref3|<nowiki>[3]</nowiki>]] This pyruvate molecule is now substrate for the ethanol production.
+blabal mechanism
 <center>
@@ Line 47: / Line 15: @@
 </center>
-==Progress==
+==References==
-''as of 2009-1018''
-We have completed the construction of both the construct variants and they are currently being tested in ''E.coli'' employing the pLac promoter. The ''Z.mobilis'' variant has been transformed to to Synechocystis and is currently growing to pick able size on plate, this takes about one week.  The Yeast construct is lagging behind the Z.mobilis variant due to some failed transformations.
+{{anchor|ref1}}
+<nowiki>[1]</nowiki> [http://www.ncbi.nlm.nih.gov/pubmed/9925577 Ethanol synthesis by genetic engineering in cyanobacteria. Deng MD, Coleman JR. <i>Appl Environ Microbiol. 1999 Feb;65(2):523-8</i>]
-After transformation to ''Synechocystis'' it takes approximately two weeks till the culture has grown to enough volume to be used in tests.
+{{anchor|ref2}}
+<nowiki>[2]</nowiki> Jeremy M. Berg, John L. Tymoczko, L. Stryer <i>Biochemistry 5th Edition; Ch.21.1 p826-838<i> 653
-[[Image:Progressbar_ethanol.png]]
+{{anchor|ref3}}
+<nowiki>[3]</nowiki> Jeremy M. Berg, John L. Tymoczko, L. Stryer <i>Biochemistry 5th Edition; Ch.16.1 p653<i>
+{{anchor|ref4}}
+<nowiki>[4]</nowiki> [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-7643 MetaCyc Reaction: 4.1.1.1]
+{{anchor|ref5}}
+<nowiki>[5]</nowiki> [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-7657 MetaCyc Reaction: 1.1.1.1]
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