Team:Calgary/Project/OSCAR/Desulfurization

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<h2>Why Remove Sulfur?</h2>
<h2>Why Remove Sulfur?</h2>
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<p>
 
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Sulfur is the third most abundant element in crude oil (Ma, 2010). When sulfur containing hydrocarbons in fuel are burned, S0<sub>2</sub> and S0<sub>3</sub> are released into the atmosphere, and are major component in air pollution, acid rain, and dry acid deposition (Reichmuth, 2000). Acid rain (rain with a pH less than 5.3) can cause acidification of aquatic and terrestrial environments, causing nutrient leeching and damaging the health and habitats of many species not tolerant to acidification. Sulfur dioxide can also aggravate respiratory health issues. In addition, sulfur in fuels can reduce the effectiveness of vehicular emmision control measures such as catalytic converters, as well as reduce the life and cause damage to valuable catalysts in the oil refining process.</p><p>
 
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Stringent regulations on sulfur content in fossil fuels have been put into place. As of 2005, low-sulfur gasoline (gasoline with a sulfur content of less than 30 mg/kg) is required across all of Canada (Source: Environment Canada). Naphthenic acid compounds found in the tailings ponds can also be sulfur-containing heterocycles. Because our aim is to convert these compounds to useable fuel, these sulfur atoms must therefore be removed from the naphthenic acids before this goal can be realized. In addition, removal of the sulfur atom in a heterocycle alters the connectivity of the carbon skeleton of the molecule, changing it from a 3-ring compound to a 2-ring compound.
 
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<h2>Our Vision</h2><p>
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<p align="justify">
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The current process of hydrodesulfurization used to remove sulfur during fuel upgrading is environmentally unsound (requiring high temperature and pressure) as well as being quite costly. This process also releases the sulfur in the form of hydrogen sulfide gas, a toxic compound which is then converted to elemental sulfur or sulfuric acid. During this process temperatures range around 350&deg;C with pressures as high as 130 atm. In contrast to this, microbes have been found capable of harvesting the sulfur out of hydrocarbons, a process which on an industrial scale would be much more environmentally sound and most likely be less costly to carry out. Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway). This is unwanted in our system, because by removing some of the carbon backbone to be turned into biomass, the cells effectively reduce the fuel quality.</p>
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Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.</p>
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<p>The pathway we have chosen for our system is not destructive in this fashion, and therefore preserves (and improves) fuel quality. The 4S pathway found in Rhodococcus spp. has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl in a non-destructive manner. DBT and its derivatives make up 60% of the organic sulfur compounds found in crude oil, and are also some of the most difficult to remove. The ability of the 4S pathway to act on derivatives of DBT leads us to hope that it will in addition be capable of acting upon naphthenic acids containing sulfur atoms in their structure.  
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<h2>Our Vision</h2><p align="justify">
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Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time.  
</p>
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|500px]]<html></p>
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p>
<h2>4S pathway</h2>
<h2>4S pathway</h2>
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<p>
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<p align="justify">
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Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of oxygen to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the heteroatom, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-hydroxybiphenyl. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p>  
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Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p align="justify">
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The first three steps of the 4S pathway carried out by the monooxygenase enzymes require FMNH<sub>2</sub>, and therefore use up the reductive power of the cell. Without a way to recover this, the pathway essentially chokes, desulfurization rates are very low, and cell health suffers. In order to fix this problem, an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed.</p><p>
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The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify">
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<i>dszA</i>, <i>dszB</i> and <i>dszC</i> form an operon on the pSOX plasmid of <i>R. erythropolis</i> while <i>dszD</i> (a flavin oxidoreductase) is present on the chromosomal DNA of the organism. In natural circumstances the 4S pathway has been found to be quite slow, preventing this method of biodesulfurization from becoming commercially feasable. This is due to a number of limitations found in the native setup of the system. With synthetic biology approaches however, it is possible to optimize this pathway by making some alterations. </p>
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The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p>
<h2>Our Approach</h2>
<h2>Our Approach</h2>
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<h3><u>1) Find an organism</h3></u>
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<a name="Degradation"></a><h3>1) Find the genes!</h3>
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<p><i>Rhodococcus spp.</i> have been shown in previous work to be capable of the desulfurization of DBT. An environmental isolate of <i>Rhodococcus</i> shown to be an active desulfurizer was obtained to be the source of the <i>dsz</i> genes we intended to biobrick. The promoter controlling the transcription of this pathway is normally repressed by sulfur containing compounds such as cysteine, methionine, and sulfate (Li et al., 1996). This first problem is easily solved, since our final construct will have a promoter from the registry instead of the native gene promotor, preventing repression from interfering with the expression of the pathway.</p><p>
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<p align="justify">We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p>
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In order to get the <i>dsz</i> genes, the desulfurization operon-containing plasmid had to be isolated from the organism. This was performed using a modified version of the miniprep procedure used on <i>Escherichia coli</i>, with the addition of lysozyme so that the cell wall of the gram-positive organism could be broken down. Primers were then designed to amplify the <i>dszA, dszB,</i> and <i>dszC</i> genes from the plasmid DNA, forward primers designed against each gene were designed. These primers contained the biobrick prefix and suffix at their tail ends in order to allow for the construction of the PCR amplicon into a standard biobrick backbone. After fine-tuning PCR conditions using gradient PCR to find conditions that would allow for the amplification of our gene out of the plasmid, each of the <i>dsz</i> genes was constructed into a pSB1C3 backbone. After these parts had been sequence confirmed, the next step would have been to construct these genes into a complete operon for desulfurization. However, through examining the sequences, it was found that before this could be done, steps of mutagenesis would have to be performed in order to remove illegal biobrick cut sites that would interfere with later construction.</p>
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<p align="justify"> We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our <i>Rhodococcus</i> strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur  containing toxins, including those that resemble naphthenic acids. </p>  
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<h3><u>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3></u>
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<p align="justify"></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation. ]]<html></p>
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<p>When sequences were examined, the native <i>dszA</i> was found to have four PstI cut sites. Similarily, <i>dszB</i> has a PstI and a NotI cut site, and <i>dszC</i> has two PstI cut sites. In order to be able to use these parts in construction and have them become part of the standard biobrick system, we had to eliminate these cut sites through site-directed mutagenesis. The protocol for Stratagene site directed mutagenesis was used in order to design our mutagenic primers, and for the basics of the entire process, however we chose to use a different (KAPA Biosystems) polymerase because it was available and because of its high fidelity. Because the procedure requires the entire length of the plasmid to be amplified through PCR, we wanted to minimize the risk of any errors being made during this process that would introduce mutations into our genes. During this procedure, two primers are designed for each mutation. Primers are complementary to the DNA strands except for one base pair where the mutation is added, and are complementary to each other so that the mutation site is in the centre of each. The mutation to the DNA was chosen in a way so that a one base pair point mutation is introduced which eliminates the cut site but the codon it belongs to still codes for the same amino acid (a silent mutation). These primers then amplify outward, and  therefore after the PCR the whole plasmid is amplified with a point mutation at the desired site.</p><p>
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<p align="justify"></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p>
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While we were designing primers to make the <i>dsz</i> genes biobrick compatible, we came across a report in the literature which found a point mutation that was capable of increasing the activity of DszB. This Y63F change to the protein structure changes a tyrosine residue to a phenylalanine. In order to screen for positive colonies resulting from a successful mutation, an HpyAV cut site was also built in without changing the amino acid sequence. The same mutagenesis procedure used for the biobrick sites was used to add this modification to our system, in order to further increase its efficiency. </p>
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<p align="justify">
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<h3><u>3) Replacing DszD with HpaC & Introducing Catalase </h3></u>
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</html>[[File:Ucalgary2012-SulfurfigureDBTandothersdegradation.png|center|800px|thumb|Figure 19: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM of the indicated compound ('''A:''' dibenzothiophene, '''B:''' tetrahydro-4h-thiopyran-4-one, and '''C:''' benzo[b]thiophene-2-carboxyaldehyde)  with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdown. Degradation is seen for DBT as well as other sulfur-containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.]]<html>
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<p>
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In the 4S pathway, FMNH<sub>2</sub> is consumed by the first three steps in the pathway carried out by DszA and DszC. When this compound is consumed, the speed of oxidation of DBT is reduced as the pathway begins to halt. A high concentration of FMNH<sub>2</sub> on the other hand produces hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) that is lethal to cell in high concentrations (Gala´n et al. 2000). </p><p>
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To overcome these problems in our system, two plans of optimization were implemented. First, the <i>hpaC</i> gene coding for another flavin reductase in <i>E. coli</i> W is used in place of the <i>dszD</i> gene which is native to the pathway. It has been shown that HpaC improves the rate of desulfurization by 7-10 times when compared to DszD (Gala´n et al. 2000). Second, a catalase gene was included in the final construct in order to take care of any excess hydrogen peroxide formed during the recycling of FMNH<sub>2</sub> and prevent stress on the cell (<a href="http://partsregistry.org/Part:BBa_K137068">KatG-LAA</a>.
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</html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|Alt text]]<html>
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</p>
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<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3>
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<p align="justify">In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro <i>et al</i>., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p>
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<a name="catalase"></a><h3>3) Replacing DszD with HpaC & Introducing Catalase </h3>
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<p align="justify">
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As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>P<sub>lacI</sub>-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p>
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<p align="justify"></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p>
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<h3>Results</h3>
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<p align="justify">To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p align="justify">
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</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of P<sub>lacI</sub> and P<sub>lacI</sub>-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p>
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<p align="justify">To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p>
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<p align="justify"> </html>
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[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of P<sub>lacI</sub>-hpaC and P<sub>lacI</sub>-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. P<sub>lacI</sub>-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the P<sub>lacI</sub>-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p>
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<p align="justify">The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>P<sub>lacI</sub>-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p>
   
   
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<h3><u>4) Optimizing Gene Order</h3></u><p>
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<a name="UBC"></a><h3>4) Optimizing Gene Order</h3>
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After the genetic elements we intended to use in the pathway were selected, more research was performed into further ways to optimize the system for maximum output and efficiency in order to overcome the issues present in the natural system. Rearranging the order of the genes in the operon has also shown to be beneficial in improving the efficiency of the 4S pathway. The natural order of genes in the operon is <i>dszABC</i>, however the relative activity of the enzymes does not reflect this pattern, as the catalytic activity of DszA:DszB:DszC is 25:1:5 respectively. Because of this, rearranging the operon to <i>dszBCA</i> produces more of the enzyme with lower activity and less of the enzyme with high activity. This is accomplished simply by having more of the less active enzyme transcribed, which translates into more enzyme being present to compensate for its lower activity (Li et al. 2008). In addition, the initiation codon of <i>dszB</i> overlaps with the termination codon of <i>dszA</i> in the natural operon, which leads to translational coupling and further reduces the amount of DszB, leading to a bottleneck in the pathway. By seperately PCR'ing out each of the genes and arranging them in this fashion in a biobrick plasmid, with each separated by an RBS site part (<a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>), this issue is also eliminated.</p><p>
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</html>[[File:DszOperonOptimize.png|center|400px|Alt text]]<html>
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<p align="justify">Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><p align="justify">
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</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html>
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</a><h2>Final Sulfur Constructs</h2>
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<p align="justify">After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p align="justify">
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</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p>
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<h2>Final Constructs</h2>
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<p align="justify">
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<p>After all of the above, we hope to develop four final constructs for our system which will allow us to test the desulfurization under a few different conditions.</p><p>
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The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K804005"><i>dszC</i></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>)  under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p>
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</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|Alt text]]<html></p><p>
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<p align="justify">This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p>
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The first two constructs have the modified <i>dsz</i> operon under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <i>hpaC</i> <br> C) With the addition of both <i>hpaC</i> and <i>katG-LAA</i></p><p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.
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</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px|Alt text]]<html></p><p>
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Because there are a large number of proteins being expressed in this system, we were concerned about the possibility of inclusion bodies forming which would essentially result in an unusable system, in addition to possible other toxic effects due to protein expression. Because of this a backup system was built, where both the optimization circuit and the <i>dsz</i> operon were under the control of the lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal. Once tuning was performed, a constitutive promotor of that strength could then be implemented to maintain optimal gene expression.</p>
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<h2>References</h2>
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<p>
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http://www.unep.org/transport/pcfv/pdf/Ethiopia-AirPollutionsulphur.pdf (Retrieved: 09/18/2012)<br>
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http://www.epa.gov/air/sulfurdioxide/ (Retrieved: 09/18/2012)<br>
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http://www.ccohs.ca/oshanswers/chemicals/chem_profiles/sulfurdi/health_sul.html (Retrieved: 09/18/2012)<br>
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</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html>
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Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p>
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<p align="justify">Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.</p>
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Latest revision as of 01:17, 27 October 2012

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Desulfurization

Why Remove Sulfur?

Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S02 and S03 gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth et al., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone.

Our Vision

Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani et al., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in Rhodococcus spp. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time.

Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.

4S pathway

Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO2) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.

The first three steps of the 4S pathway require FMNH2 and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH2, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.

The dszA,B, and C genes form an operon on the pSOX plasmid of R. erythropolis, while dszD is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.

Our Approach

1) Find the genes!

We isolated the plasmid containing the dsz genes from a desulfurising environmental isolate of Rhodococcus using a modified miniprep procedure. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li et al., 1996), we designed primers for just the coding sequences of the A, B, and C genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our mutagenesis protocol.

We performed an experiment to measure the desulfurization rate of select organosulfur compounds by our Rhodococcus strain (Figures 4-6 below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the dsz operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids.

Figure 2: Rhodococcus cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through the GC/MS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GC/MS after 6 days of incubation.

Figure 3: The peak in this mass spectrum demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).

Figure 19: Rhodococcus cells were grown in a modified M9 media containing 0.125mM of the indicated compound (A: dibenzothiophene, B: tetrahydro-4h-thiopyran-4-one, and C: benzo[b]thiophene-2-carboxyaldehyde) with no other sulfur containing compounds present in the media (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of compound remaining. Samples were normalized to a control containing modified M9 but no bacteria, run through the GCMS at the last time point to account for abiotic breakdown. Degradation is seen for DBT as well as other sulfur-containing compounds resembling naphthenic acids, indicating that the pathway may have wider substrate specificity than previously thought.

2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity

In total the dszABC genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. In addition, a point mutation of Y63F in DszB increased the activity of the protein (Oshiro et al., 2007), and was included in the mass mutagenesis we undertook. Mutagenesis was performed as described in this protocol.

3) Replacing DszD with HpaC & Introducing Catalase

As FMNH2 is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the dszD gene, however it has been shown that the hpaC gene from E. coli performs the same function more efficiently (Gala´n et al., 2000). One problem arises from this though, as high levels of FMNH2 cause the production of toxic hydrogen peroxide inside the cell (Gala´n et al. 2000). To address this issue we have included a catalase gene ( PlacI-katG-LAA) that will remove the peroxide that would be toxic to the cell.

Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).

Results

To show that catalase activity increased E. coli survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.

Figure 8: Catalase Assay. Overnight cultures of PlacI and PlacI-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.

To test the action of HpaC to use NADH to recycle FMN into FMNH2 cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali et al., 2010) was measured over time. Both native HpaC expression and an induced PlacI-RBS-hpaC system were tested as well as a negative control. The results are shown below.

Figure 9: HpaC Assay with A) 2 mL cell lysate and B) 100 µL cell lysate. Cultures of PlacI-hpaC and PlacI-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 µM IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 µM NADH and 20 µM FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. PlacI-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the PlacI-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.

The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the PlacI-RBS-hpaC was functional and that it would effectively recycle FMN.

4) Optimizing Gene Order

Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li et al., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, B0034.

Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.

Final Sulfur Constructs

After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.

Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG

The first two constructs have the modified dsz operon (dszB, dszC, dszA) under the control of a constitutive TetR promotor (BBa_J13002) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (BBa_J04500). The set-up of these two constructs will therefore allow for the expression of the dsz genes with the ability to test and compare their desulfurization rates
A) On their own
B) With the addition of hpaC
C) With the addition of both hpaC and katG-LAA

This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.

Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.

Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the dsz operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.

Currently the final steps of construction of these constructs is underway, following which functionality tests will begin.