Team:Washington/Alkanes/Future/LuxCDE

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=Alternative Routes for Fatty Aldehyde Production=
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='''<center>Alternative Routes for Fatty Aldehyde Production'''=
==Background==
==Background==
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LuxCDE is an enzyme complex that produces a tetradecanal (14 carbon aldehyde) substrate by drawing from the fatty acid biosynthesis pathway. This tetradecanal substrate is to be catalyzed by the luxAB luciferase complex to produce light. The waste from the lucerferase is then sent back to luxCDE to be recycled. Since the aldehyde decarbonalse (ADC) uses aldehyde substrate to make alkane, we plan to use the luxCDE complex and ADC to synthesize alkane. The entire lux operon was submitted to the registry last year by [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases Cambridge 2010].
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[[File:Washington_luxcde_diagram.png|right|300px|thumb|A diagram of the LuxCDE pathway, producing tetradecanal, the aldehyde substrate for use with aldehyde decarbonalase (ADC)]]
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LuxABCDE is an enzyme complex submitted to the iGem registry by the 2010 Cambridge team. Within this complex, LuxAB produces luciferase (used by [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases Cambridge's] EGlowi) using 14-carbon aldehydes (tetradecanal) produced by LuxCDE. Since the aldehyde decarbonylase (ADC) in our project uses aldehydes to synthesize alkanes, we plan to use the LuxCDE complex in conjunction with ADC to produce 13-carbon alkanes (Tridecane).
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<br>
==Methods==
==Methods==
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<big>PCR</big>
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===PCR and Standard Biobrick Assembly===
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This method revolved around designing oligos to PCR amplify LuxCD and LuxE off of the LuxBrick; LuxE was not directly adjacent to LuxCD in the Luxbrick, and therefore had to be obtained separately. Sticky ends on the oligos would then allow us to obtain a LuxCDE using stitching PCR. We would then digest and cut the resulting fragment, ligate it into a similarly-cut vector, and use transformation to obtain the desired plasmid with LuxCDE embedded into it.
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This method revolved around designing oligos to PCR amplify LuxCD and LuxE off of the LuxBrick; LuxE was not directly adjacent to LuxCD in the Luxbrick, and therefore had to be amplified out in two separate reactions. Homology on the ends of the two fragments would allow us to obtain a LuxCDE using stitching PCR, then ligate this fragment and ADC into psb1C3. We would then assay for alkanes.
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<big>Gibson Assembly (mostly accurate, need to make sure on some stuff)</big>
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<br>
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We would design oligos to amplify C, D, and E off of the LuxBrick. Unlike the previous method, the oligos would have sticky ends of [    ] base pairs to prevent misannealing, and the fragment containing C would also contain the vector for ligation. A 3-piece Gibson assembly was then done on C, D, and E, and the product was then transformed into cells.
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<big>Gene Synthesis</big>
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===Gibson Assembly===
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Instead of using the LuxBrick to obtain C, D, and E, we would optimize C, D, and E, then assemble them ourselves. PCR was then used to amplify the respective pieces, and each of the three pieces was then digested and transformed into cells.
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[Still working on this, will be expanded after experiments are complete]
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[[File:Washington2011 GibsonReaction.png|right|300px|thumb|A basic diagram of a Gibson reaction. All components of the desired vector are left to ligate simultaneously, then transformed into cells.]]
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We would design oligos with homology to amplify C, D, and E off of the LuxBrick. The homology would allow the enzymes in the Gibson reaction to chew back the homologous ends and ligate them togather. A 4-piece Gibson assembly was done on C, D, E, and the plasmid backbone with along with aldehyde decarbonylase and the product was then transformed into cells. Another reaction we tried was to amplify everything on the LuxBrick except luxA and luxB. In this reaction we then switched the two genes with ADC to create a product with luxC, luxD, luxE, luxG and ADC.
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<br>
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===Gene Synthesis===
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Instead of using the LuxBrick to obtain C, D, and E, C, D, and E was re-optimized to have cloning-friendly GC content. Primers were designed to synthesize the genes separately each in their own PCR reaction. Another PCR was used to amplify the respective pieces, and each piece was ligated into psb1C3. Once each gene was in psb1C3, it was re-amplified out and ligated into a vector containing ADC. This is done step wise until the full construct is obtained.
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<br>
==Results==
==Results==
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The PCR and Gibson methods failed at multiple steps for uncertain reasons. Since we do not have our LuxCDE construct, no method or assay for testing the hypothetical construct was developed as of this time. We hypothesize that LuxCD's abnormally high AT concentration may have caused misannealing for primers, causing primer-dimer errors and other problems, but no concrete evidence exists for this as of yet. For gene assembly, we have lowered the AT concentration of C, D, and E to more optimal levels for PCR.
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The PCR and Gibson methods failed at multiple steps for uncertain reasons. Since we do not have our LuxCDE construct, no method or assay for testing the hypothetical construct was developed as of this time. We hypothesize that LuxCD's abnormally high AT concentration may have caused misannealing for primers, causing primer-dimer errors and other problems, but no concrete evidence exists for this as of yet. For gene assembly, we have lowered the AT concentration of C, D, and E to more optimal levels for PCR. To be precise, the C, D, and E had an AT content of 66.3, 65.0, and 66.9 percent, which was reduced to 50.2, 50.1, and 51.6 percent respectively. Re-optimization likely helped cleanly amplifying out the genes.
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[Still working on this, will be expanded once all experiments are complete]
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<br>
==Current Status==
==Current Status==
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Attempts to clone LuxCDE using the PCR and Gibson method have been abandoned. Gene assembly is still a work in progress; sequencing was being done at the time of writing.
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Attempts to clone LuxCDE using standard PCR/Biobrick Assembly and Gibson Assembly have been abandoned. Gene assembly was successful and each of the three luxCDE genes have been sequenced, biobricked and submitted. We are currently working on assembling the full CDE + ADC construct.
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==Parts Submitted==
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<html>
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This is also a work in progress, seeing we haven't completed our experiments yet.
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<img src=https://static.igem.org/mediawiki/2011/e/eb/Washington_cde_construct.png width="750" height="353" usemap="#washington cde construct.png"/>
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<map name="washington cde construct.png">
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<area shape="rect" coords="143,99,294,250" title="Part:BBa_K590031" href="http://partsregistry.org/Part:BBa_K590031"/>
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<area shape="rect" coords="48,107,143,201" title="Part:BBa_J23100" href="http://partsregistry.org/Part:BBa_J23100"/>
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<area shape="rect" coords="294,99,446,250" title="Part:BBa_K590059" href="http://partsregistry.org/Part:BBa_K590059"/>
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<area shape="rect" coords="446,99,598,250" title="Part:BBa_K590060" href="http://partsregistry.org/Part:BBa_K590060"/>
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<area shape="rect" coords="598,99,750,250" title="Part:BBa_K590061" href="http://partsregistry.org/Part:BBa_K590061"/>
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</map>
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</html>
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==Image(s)==
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==Parts Submitted==
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590059 LuxC], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590060 LuxD], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590061 LuxE]
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Subsection will be deleted and intergrated into article once images are found.
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Latest revision as of 02:22, 29 September 2011


Alternative Routes for Fatty Aldehyde Production

Background

A diagram of the LuxCDE pathway, producing tetradecanal, the aldehyde substrate for use with aldehyde decarbonalase (ADC)

LuxABCDE is an enzyme complex submitted to the iGem registry by the 2010 Cambridge team. Within this complex, LuxAB produces luciferase (used by Cambridge's EGlowi) using 14-carbon aldehydes (tetradecanal) produced by LuxCDE. Since the aldehyde decarbonylase (ADC) in our project uses aldehydes to synthesize alkanes, we plan to use the LuxCDE complex in conjunction with ADC to produce 13-carbon alkanes (Tridecane).


Methods

PCR and Standard Biobrick Assembly

This method revolved around designing oligos to PCR amplify LuxCD and LuxE off of the LuxBrick; LuxE was not directly adjacent to LuxCD in the Luxbrick, and therefore had to be amplified out in two separate reactions. Homology on the ends of the two fragments would allow us to obtain a LuxCDE using stitching PCR, then ligate this fragment and ADC into psb1C3. We would then assay for alkanes.


Gibson Assembly

A basic diagram of a Gibson reaction. All components of the desired vector are left to ligate simultaneously, then transformed into cells.

We would design oligos with homology to amplify C, D, and E off of the LuxBrick. The homology would allow the enzymes in the Gibson reaction to chew back the homologous ends and ligate them togather. A 4-piece Gibson assembly was done on C, D, E, and the plasmid backbone with along with aldehyde decarbonylase and the product was then transformed into cells. Another reaction we tried was to amplify everything on the LuxBrick except luxA and luxB. In this reaction we then switched the two genes with ADC to create a product with luxC, luxD, luxE, luxG and ADC.


Gene Synthesis

Instead of using the LuxBrick to obtain C, D, and E, C, D, and E was re-optimized to have cloning-friendly GC content. Primers were designed to synthesize the genes separately each in their own PCR reaction. Another PCR was used to amplify the respective pieces, and each piece was ligated into psb1C3. Once each gene was in psb1C3, it was re-amplified out and ligated into a vector containing ADC. This is done step wise until the full construct is obtained.


Results

The PCR and Gibson methods failed at multiple steps for uncertain reasons. Since we do not have our LuxCDE construct, no method or assay for testing the hypothetical construct was developed as of this time. We hypothesize that LuxCD's abnormally high AT concentration may have caused misannealing for primers, causing primer-dimer errors and other problems, but no concrete evidence exists for this as of yet. For gene assembly, we have lowered the AT concentration of C, D, and E to more optimal levels for PCR. To be precise, the C, D, and E had an AT content of 66.3, 65.0, and 66.9 percent, which was reduced to 50.2, 50.1, and 51.6 percent respectively. Re-optimization likely helped cleanly amplifying out the genes.


Current Status

Attempts to clone LuxCDE using standard PCR/Biobrick Assembly and Gibson Assembly have been abandoned. Gene assembly was successful and each of the three luxCDE genes have been sequenced, biobricked and submitted. We are currently working on assembling the full CDE + ADC construct.

Parts Submitted

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590059 LuxC], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590060 LuxD], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590061 LuxE]
Retrieved from "http://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE"