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<center>[https://2007.igem.org/Virginia http://www.parking-net.com/Upload/Jobs/6162/university-of-va-web-logo.jpg]</center>
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[[Image:UVA Logo.gif|center]]
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=='''Biosynthesis of butanol biofuel by a synthetic metabolic pathway in ''E. coli'''''==
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We have designed a metabolic system capable of cellulose degradation and light metabolism in order to power the biosynthesis of butanol fuel.  This hybrid molecular engine is built from standard biological parts and may be used in future designs in order to drive forward cellular chemistry.
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The coming years are going to require us to revamp our notions of a fuel economy. Our team hopes to show how
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{| cellspacing="6px" cellpadding="16" border="0" width="100%"
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synthetic biology can aid in tackling real-world problems not only to aid in the development of new fuel
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|-
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technologies, but also to help support the synthetic biology community by showing it's utility.
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|[[Virginia|HOME]]
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|[[Virginia/Projects|PROJECT INTRO]]
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|[[Virginia/Projects/1|APPROACH]]
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|[[Virginia/Projects/2|PROCEDURES]]
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|[[Virginia/Projects/3|RESULTS]]
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|[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2007&group=Virginia BIOBRICKS]
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|[http://openwetware.org/wiki/IGEM:VGEM/2007/Notebook eNOTEBOOK]
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|[http://www.seas.virginia.edu/VGEM/ WEBSITE]
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|}
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==Harvesting Cellulose and Light to Power Butanol Biosynthesis==
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Send comments to [mailto:mcarthur@virginia.edu?subject=VGEM_Query George McArthur]
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===Background, Motivation, and References===
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===Approach===
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----
 +
New approaches to metabolic engineering made possible by the development of genomics, systems biology, and synthetic biology have enabled metabolic pathway design in microorganisms for the production of transportation fuels such as ethanol, butanol and hydrogen (Stephanopoulos 2007).  The novel microbial metabolism developed by the 2007 Virginia Genetically Engineered Machine (VGEM) Team for the production of butanol from cellulose and light is illustrated below in Figure 1.  The four main systems that comprise the pathway- cellulose metabolism, light metabolism, butanol biosynthesis, and butanol tolerance- have extended E. coli K-12’s natural central metabolic pathway.  In this way, a valuable chemical product such as butanol biofuel can be biologically manufactured using inexpensive feedstock material.  The cellulose and light metabolizing systems may one day serve as engines in future system designs to drive forward the biosynthesis of chemical products in microorganisms.
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<br />
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<br />
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<center>[[Image:VGEM 2007 metabolic pathway.jpg|800px]]</center>
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<br />
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<center>'''Figure 1'''</center>
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<br />
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The cellulase system was designed using three cellulases discovered in Saccharophagus degradans.  Cel5F, Cel6A, and Bgl3C have been predicted to code for an endo-1,4,-β-glucanase, cellobiohydrolase, and cellobiase respectively (Taylor et al. 2006).  These enzymes are capable of converting crystalline cellulose to long cellulose chains, then to cellobiose units containing two or four β-glucose monomers, and finally to β-glucose units that can be taken up by E. coli.  Cellulose is a complex polysaccharide that is too large to be transported to the inside of the cell.  Therefore, the three enzymes must be secreted to act on the cellulose substrate.  According to Taylor et al., these three genes include a type II secretion signal sequence, which should take advantage of the type II secretion system that is native to E. coli.
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'''Why Butanol? '''  
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To aid in powering E. coli growth and butanol production, proteorhodopsin was incorporated into the pathway design.  Proteorhodopsin re-establishes the proton motive force that is decreased under non-ideal conditions such as low glucose concentrations or respiratory stress (Walter et al., 2006)).  Butanol biosynthesis occurs under anaerobic conditions naturally in Clostridia.
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<br />
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<br />
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<center>[[Image:VGEM 2007 BioBrick table.jpg|800px]]</center>
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<br />
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<center>'''Table 1'''</center>
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<br />
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The ten genes that were used to design the system, listed above in Table 1 in BioBrick nomenclature, were standardized to facilitate system construction via BioBrick assembly methods.  Each BioBrick was designed by combining the gene of interest (i.e., the protein coding region) with other basic parts including a promoter (BBa_J23119), a ribosomal binding site (BBa_B0030), and two terminators (BBa_0012 and BBa_0011).  The construct is defined as a BioBrick once it includes a standard prefix (before the promoter) and a standard suffix (after the terminator) that code for restriction enzyme sites.  EcoRI, NotI, and XbaI are sites contained in the prefix while SpeI, NotI, and PstI are included in the suffix sequence.
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As energy demands increase, the need for alternative fuel sources increases dramatically.  US market size for butanol: 370 million gallons per year at a price of about $3.75 per gallon. That’s $1.4 billion.
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The experimental design was determined by the metabolic pathway and the fact that more complex BioBrick systems can be assembled from simpler ones.  The modularity of BioBricks can be taken full advantage of in this project by taking a bottom-up approach to pathway design. For example, butanol dehydrogenase (BBa_I711021) may or may not be a necessary enzyme for the production of butanol (6).  That is, the butyraldehyde dehydrogenases in the prior enzymatic step may actually be responsible for both steps.  To test this, we designed an experiment that compares the fermentation product distribution of E. coli that would be transformed with both butyraldehyde dehydrogenases and butanol dehydrogenase to E. coli that would be transformed with only the butyraldehyde dehydrogenases.
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'''What is it used for?'''
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Chemical and textile solvent, organic synthesis, chemical intermediate, paint thinner, base of perfumes, and, most importantly, as a biofuel.   
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The detailed schematic shown below (Figure 2) demonstrates this powerful approach to biological system design and construction.  As simpler systems are tested and characterized, they are consolidated into more complex systems.  Eventually, the four main systems that make up this pathway would become abstracted.  This is the goal.  At that point, one could look at the entire metabolic pathway as a black box with cellulose and light going in and butanol and waste products coming out.
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<br />
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'''As a Biofuel'''
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<br />
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<center>[[Image:VGEM 2007 biobrick construction strategy.jpg|800px]]</center>
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Butanol biofuel can be used in cars without making engine modifications.  It produces more power than ethanol and almost as much power as gasoline.
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<br />
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<center>'''Figure 2'''</center>
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Butanol better tolerates water contamination and is less corrosive than ethanol and more suitable for distribution through existing pipelines for gasoline.
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<br />
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===References===
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'''Why isn’t it more widely spread?'''
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Historically low yields and low concentrations of biobutanol when compared to bioethanol have prevented industry from having stronger interest.   
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Product tolerance is the main issue.  Butanol-producing bacteria (''Clostridia acetobutylicum'') become limited in growth at approximately 2.5% concentration.  Isolating the product at this concentration is not economical.
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In the 1950s butanol production shifted from fermentation to being petrochemically-derived.  This method continues to be the most popular today. 
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There are developments in biobutanol production, however.  Recently BP and Dupont announced the conversion of an ethanol fermentation facility in the UK to a dedicated biobutanol plant.  Biobutanol from this plant will be introduced in 2007.
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'''References:'''
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http://www.butanol.com/
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http://www.greencarcongress.com/2006/06/bp_and_dupont_t.html
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http://i-r-squared.blogspot.com/2006/05/bio-butanol.html
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http://en.wikipedia.org/wiki/Butanol
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===Our Project===
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----
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Our goal is to isolate the pathway of butanol production existing in various organisms and engineering the metabolic pathway of E.Coli to produce butanol.  Butanol limits bacterial growth by degrades cellular membranes, so the first step is to convey butanol tolerance to E.Coli.  This will be accomplished via the use of tolerance genes from other bacterial species. 
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Next, we will transform the cells with the necessary enzymes for butanol production.  These are explained in detail below.  By growing the cells in anaerobic conditions and analyzing their product, we hope to tweak the pathway to produce maximum amounts of butanol.
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One approach to this is to vary the energy sources the bacteria can use.  By inserting genes coding for cellulase, we hope to give our cells the ability to use cellulose as an energy source.  Agricultural waste would then become the feed for our strains.  Additionally, the use of proteorhodopsin to supplement ATP production is planned.  Proteorhodopsin allow the cells to harness light energy independent of oxygen in the environment and drive cellular metabolism. 
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Our final goal is to design a system that will allow E.Coli to be tolerant to butanol, produce butanol, and do so by exploiting various energy sources to increase efficiency and large-scale feasibility.
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===Biobrick and pathway design===
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----
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[[Image:untitled.jpg]]
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Image source: http://jb.asm.org/cgi/content/full/183/16/4823/F5
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This is the pathway we are incorporating into E.Coli.
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Clostridium acetbutylicum has been known to produce butanol anaerobically in nature. For our project we will be
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utilizing two butanol sythesis pathways available to us from its genome. In addition to this, we plan to provide a
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carbon source for butanol biosythesis by importing a cellulase gene from Saccaruphagus degradans, and we plan to
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facilitate the proton gradient required for ATP sythesis through the use of a proteorhodopsin system.
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The diagram below depicts the complete system that we plan to create. The added pathways are: 3, 4, 5, 6, 9, 10,
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11. Excluded from this image is our plans for the cellulose digestion pathway, which essentially feed into the
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production of pyruvate.
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The specific proteins we need the cells to produce for this pathway are:
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Specific cellulase,
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butanol tolerance genes,
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thiolase,
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beta-hydroxybutyryl-CoA dehydrogenase,
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crotonase, 
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butyryl coa dehydrogenase
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AAD/AAD2
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alcohol dehydrogenase,
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AOTC/AOTD
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'''We have designed the following biobricks:'''
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Need this info.
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===Methods and Materials===
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----
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We will analyze butanol production via gas chromatography and mass spectroscopy.
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Our experimental scaffold (still in development) is as follows:
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What is the butanol tolerance of WT cells and of transformed cells?
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1) Butanol Tolerance
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Hypothesis: Transformed cells will be more resistant to extracellular butanol than WT
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    * Add varying amounts of butanol to broths of transformed and non-transformed cells
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    * Determine amounts of butanol cells can withstand before dying
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    * Count cells with hemocytometer and blue stain
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Note:  Depending on butanol tolerance we will consider butanol tolerance transformed cells as wildtype (WT).  If it doesn't give any significant tolerance we will just use the original strain.
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Can cells survive with only cellulose carbon source?
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2) Effect of Cellulase
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Hypothesis:  Transformed cells should be able to grow on low glucose/high cellulose media.  No growth for WT.
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    * Transform cells with cellulase
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    * Grow transformed cells and control cells on low glucose media supplemented with cellulose
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    * Observe growth amounts
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Does thiolase increase the production of acetoacetyl-CoA from acetyl-CoA relative to WT?
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3) Effect of Thiolase
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Hypothesis:  Thiolase-transformed cells will have increased acetoacetyl-CoA production than WT, and most if cellulase-transformed
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    * Transform cells with thiolase
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    * Transform cellulase-cells (ex.2) with thiolase
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    * Plate transformed cells (thiolase and thiolase + cellualse, and control cells)
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    * Test for acetoacetyl-CoA production
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Note: If thiolase confers much greater acetoacetyl-CoA production, we will include it by default in the rest of the experiments requiring the central pathway.
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How much butanol is produced from acetyl coa to butanol, with and without alcohol dehydrogenase?
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4a) Acetyl CoA to Butanol  (-cellulase, +thiolase, +central pathway, +aad, -alcohol dehydrogenase, -AOTC/AOTD)
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Hypothesis:  Transformed cells will produce butanol.  WT will not
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    * Transform cells with thiolase, central pathway, aad/aad2
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    * Grow transformed and control cells
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    * Test for Butanol
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4b) Acetyl CoA to Butanol (-cellulase, +thiolase, +central pathway, +aad, +alcohol dehydrogenase, -AOTC/AOTD)
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Hypothesis:  Transformed cells will produce butanol, more than in ex. 4a.  WT will not
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    * Transform cells with thiolase, central pathway, aad/aad2, alcohol dehydrogenase
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    * Test for butanol production
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How much butanol is produced from cellulase to acetyl coa to butanol, with and without alcohol dehydrogenase?
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5a)Cellulase to Acetyl CoA to Butanol (+cellulase, +thiolase, +central pathway, +aad, -alcohol dehydrogenase, -AOTC/AOTD)
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Hypothesis: Transformed cells will produce butanol.  WT will not.
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    * Transform cells with cellulase, thiolase, central pathway, and aad/aad2
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    * Grow transformed and control cells
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    * Test for Butanol
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5b) Cellulase to Acetyl CoA to Butanol (+cellulase, +thiolase, +central pathway, +aad, +alcohol dehydrogenase, -AOTC/AOTD)
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Hypothesis: Transformed cells will produce butanol, and at higher amounts than in ex. 5a.  WT will not.
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    * Transform cells with cellulase, thiolase, central pathway, aad/aad2, alcohol dehydrogenase
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    * Grow transformed and control cells
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    * Test for Butanol
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How much butanol is produced via only the bottom pathway (AOTC/AOTD genes) with and without the alcohol dehydrogenase?
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6a)Butyrate (butyric acid) to Butanol (-cellulase, -thiolase, -central pathway, +aad, -alcohol dehydrogenase, +AOTC/AOTD) IN FLOW
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Hypothesis:  Pathway will produce butanol.  There will be no butanol in WT
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    * Transform cells with AOTC/AOTD and aad/aad2
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    * Grow transformed and control cells
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    * Measure butanol production with varying concentrations of initial butyric acid
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6b) Butyrate (butyric acid) to Butanol (-cellulase, -thiolase, -central pathway, +aad, +alcohol dehydrogenase, +AOTC/AOTD)
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Hypothesis:  Pathway will produce butanol, more than in ex.6a.  There will be no butanol in WT
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    * Transform cells with aad, AOTC/AOTD, and alc. dehydro.
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    * Grow transformed and control cells
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    * Measure butanol production with varying concentrations of initial butyric acid
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How much butanol from acetyl coa to butanol, including AOTC/AOTD, and with and without alcohol dehydrogenase?
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7a) Acetyl CoA to Butanol with Butyrate pathway (-cellulase, +thiolase, +central pathway, +aad, -alcohol dehydrogenase, +AOTC/AOTD)
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Hypothesis:  This is the complete pathway, but without alcohol dehyd.  High butanol production expected.
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    * Transform cells with thiolase, central pathway, aad, AOTC/AOTD
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    * Test for butanol production
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7b) Acetyl CoA to Butanol with Butyrate pathway (-cellulase, +thiolase, +central pathway, +aad, +alcohol dehydrogenase, +AOTC/AOTD)
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Hypothesis:  This is the complete pathway, without cellulase.  High butanol production expected.
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    * Transform cells with thiolase, central pathway, aad, AOTC/AOTD, and alc. dehydro.
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    * Test for butanol production
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How much butanol from cellulase to acetyl coa to butanol, including ctfa/b, and with and without alcohol dehydrogenase?
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8a) Cellulase to Butanol with Butyrate pathway (+cellulase, +thiolase, +central pathway, +aad, -alcohol dehydrogenase, +AOTC/AOTD)
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Hypothesis:  This is the complete pathway, without alcohol dehydr.  There should be maximum production of butanol.
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    * Transform cells with cellulase, thiolase, central pathway, aad, AOTC/AOTD, and alc. dehydro.
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    * Test for butanol production
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8b) Cellulase to Butanol with Butyrate pathway (+cellulase, +thiolase, +central pathway, +aad, +alcohol dehydrogenase, +AOTC/AOTD)
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Hypothesis:  This is the complete pathway.  There should be maximum production of butanol.
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    * Transform cells with cellulase, thiolase, central pathway, aad, AOTC/AOTD, and alc. dehydro.
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    * Test for butanol production
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Will cells transformed with the proteorhodopsin pathway survive with only light, will it produce butanol, and if so how much will it increase butanol production when combined with other pathways?
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9a) Effect of Proteorhodopsin
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    * Transform cells with proteorhodopsin genes
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    * grow on minimal nutrient media
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    * observe growth vs. control strains
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9b) Proteorhodopsin to butanol
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    * If ex.9a cells grow at all,
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    * transform cells with central pathway genes and proteorhodopsin
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    * test for butanol production
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9c) Proteorhodopsin to butanol, full pathway
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    * add proteorhodopsin to ex.8b cells
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    * test for butanol production
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===Results and Conclusions===
+
----
----
-
TBD
+
#Andrykovitch, G., & Marx, I. (1988). Applied and Environmental Microbiology, 54(4), 1061-1062.
 +
#Beja, O., Aravind, L., Koonin, E., Suzuki, M., Hadd, A., Nguyen, L., et al. (2000). Science, 289(5486), 1902-#906.
 +
#Borden, J., & Papoutsakis, E. (2007). Applied and Environmental Microbiology, 73(9), 3061-3068.
 +
#Kennedy, J., Murli, S., & Kealey, J. T. (2003). Biochemistry, 42(48), 14342-14348.
 +
#Martinez, A., Bradley, A. S., Waldbauer, J. R., Summons, R. E., & DeLong, E. F. (2007). Proceedings of the National Academy of Sciences, 104(13), 5590-5595.
 +
#Nolling, J., Breton, G., Omelchenko, M., Makarova, K., Zeng, Q., Gibson, R., et al. (2001). The Journal of Bacteriology, 183(16), 4823-4838.
 +
#Taylor, L.,II, Henrissat, B., Coutinho, P., Ekborg, N., Hutcheson, S., & Weiner, R. (2006). The Journal of Bacteriology, 188(11), 3849-3861.
 +
#Schwarz, W.H. (2001). Appl. Microbiol. Biotechnol. 56, 634–649
 +
#Demain, A., Newcomb, M., & Wu, J. H. D. (2005) Microbiology and Molecular Biology Reviews, 69(1), 124-154.
 +
#Ekborg, N., Gonzalez, J., Howard, M., Taylor, L., Hutcheson, S., & Weiner, R. (2005). International Journal of Systematic and Evolutionary Microbiology, 55(4), 1545-1549.
 +
#Stephanopoulos, G. (2007). Science, 315(5813), 801-804.
 +
#Walter, J., Greenfield, D., Bustamante, C., & Liphardt, J. (2007). Proceedings of the National Academy of Sciences, 104(7), 2408-2412.

Latest revision as of 05:23, 27 October 2007

UVA Logo.gif
HOME PROJECT INTRO APPROACH PROCEDURES RESULTS [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2007&group=Virginia BIOBRICKS] [http://openwetware.org/wiki/IGEM:VGEM/2007/Notebook eNOTEBOOK] [http://www.seas.virginia.edu/VGEM/ WEBSITE]

Harvesting Cellulose and Light to Power Butanol Biosynthesis

Send comments to George McArthur

Approach

New approaches to metabolic engineering made possible by the development of genomics, systems biology, and synthetic biology have enabled metabolic pathway design in microorganisms for the production of transportation fuels such as ethanol, butanol and hydrogen (Stephanopoulos 2007). The novel microbial metabolism developed by the 2007 Virginia Genetically Engineered Machine (VGEM) Team for the production of butanol from cellulose and light is illustrated below in Figure 1. The four main systems that comprise the pathway- cellulose metabolism, light metabolism, butanol biosynthesis, and butanol tolerance- have extended E. coli K-12’s natural central metabolic pathway. In this way, a valuable chemical product such as butanol biofuel can be biologically manufactured using inexpensive feedstock material. The cellulose and light metabolizing systems may one day serve as engines in future system designs to drive forward the biosynthesis of chemical products in microorganisms.

VGEM 2007 metabolic pathway.jpg


Figure 1


The cellulase system was designed using three cellulases discovered in Saccharophagus degradans. Cel5F, Cel6A, and Bgl3C have been predicted to code for an endo-1,4,-β-glucanase, cellobiohydrolase, and cellobiase respectively (Taylor et al. 2006). These enzymes are capable of converting crystalline cellulose to long cellulose chains, then to cellobiose units containing two or four β-glucose monomers, and finally to β-glucose units that can be taken up by E. coli. Cellulose is a complex polysaccharide that is too large to be transported to the inside of the cell. Therefore, the three enzymes must be secreted to act on the cellulose substrate. According to Taylor et al., these three genes include a type II secretion signal sequence, which should take advantage of the type II secretion system that is native to E. coli.

To aid in powering E. coli growth and butanol production, proteorhodopsin was incorporated into the pathway design. Proteorhodopsin re-establishes the proton motive force that is decreased under non-ideal conditions such as low glucose concentrations or respiratory stress (Walter et al., 2006)). Butanol biosynthesis occurs under anaerobic conditions naturally in Clostridia.

VGEM 2007 BioBrick table.jpg


Table 1


The ten genes that were used to design the system, listed above in Table 1 in BioBrick nomenclature, were standardized to facilitate system construction via BioBrick assembly methods. Each BioBrick was designed by combining the gene of interest (i.e., the protein coding region) with other basic parts including a promoter (BBa_J23119), a ribosomal binding site (BBa_B0030), and two terminators (BBa_0012 and BBa_0011). The construct is defined as a BioBrick once it includes a standard prefix (before the promoter) and a standard suffix (after the terminator) that code for restriction enzyme sites. EcoRI, NotI, and XbaI are sites contained in the prefix while SpeI, NotI, and PstI are included in the suffix sequence.

The experimental design was determined by the metabolic pathway and the fact that more complex BioBrick systems can be assembled from simpler ones. The modularity of BioBricks can be taken full advantage of in this project by taking a bottom-up approach to pathway design. For example, butanol dehydrogenase (BBa_I711021) may or may not be a necessary enzyme for the production of butanol (6). That is, the butyraldehyde dehydrogenases in the prior enzymatic step may actually be responsible for both steps. To test this, we designed an experiment that compares the fermentation product distribution of E. coli that would be transformed with both butyraldehyde dehydrogenases and butanol dehydrogenase to E. coli that would be transformed with only the butyraldehyde dehydrogenases.

The detailed schematic shown below (Figure 2) demonstrates this powerful approach to biological system design and construction. As simpler systems are tested and characterized, they are consolidated into more complex systems. Eventually, the four main systems that make up this pathway would become abstracted. This is the goal. At that point, one could look at the entire metabolic pathway as a black box with cellulose and light going in and butanol and waste products coming out.

VGEM 2007 biobrick construction strategy.jpg


Figure 2


References

  1. Andrykovitch, G., & Marx, I. (1988). Applied and Environmental Microbiology, 54(4), 1061-1062.
  2. Beja, O., Aravind, L., Koonin, E., Suzuki, M., Hadd, A., Nguyen, L., et al. (2000). Science, 289(5486), 1902-#906.
  3. Borden, J., & Papoutsakis, E. (2007). Applied and Environmental Microbiology, 73(9), 3061-3068.
  4. Kennedy, J., Murli, S., & Kealey, J. T. (2003). Biochemistry, 42(48), 14342-14348.
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