Difference between revisions of "Team:CCA San Diego/Description"

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CCA_San_Diego has isolated and modified two major PAH degradation pathways: fluorene and phenanthrene, in order to further understand the scope, efficiency, and application of these pathways for commercial use. These isolated systems serve to limit unwanted bacterial byproducts by allowing the selection of specific strains for expression, to allow for selection of optimal bacteria in field situations, and to reduce the use of harmful bacterial strains that contain many of the pathways we isolated.
 
CCA_San_Diego has isolated and modified two major PAH degradation pathways: fluorene and phenanthrene, in order to further understand the scope, efficiency, and application of these pathways for commercial use. These isolated systems serve to limit unwanted bacterial byproducts by allowing the selection of specific strains for expression, to allow for selection of optimal bacteria in field situations, and to reduce the use of harmful bacterial strains that contain many of the pathways we isolated.
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<h3>OUR SOLUTION</h3>
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Ocean Crude Oil Pump and Treatment
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We proposed a novel method of degradation of multiple PAHs through combinational implementation of these bacteria-derived pathways into E. coli. Thus, this treatment allows for a broad spectrum transformation of multiple PAHs within the same oil environment into safer residues. Isolation of the bacteria allows for its implementation into a spill site or in bioreactors, achieving efficient detoxification through combination genetic bioremediation.
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CCA_San_Diego designed bacteria to aid in the process of bioremediation for pump and treat ex-situ methods. Once spills are identified, contaminated sites may be pumped to remediation centers that contain our synthetic PAH-degredetory bacteria. Please see our applied design page for more information about this process.  
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The degradation pathway of each of these PAHs begins with oxygenation, followed by degradation of the aromatic ring. Each of these pathways includes a complex set of 10-15 genes typically organized in operons, encoding different classes of enzymes, mainly oxygenase, hydrogenase, and carboxylase. Additional genes are involved in the regulation of expression of the pathway (activator), secreting surfactants to allow the bacteria to mix well with crude oil, and the transport of PAHs inside the microorganism where the degradation takes place.  
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<img src="https://static.igem.org/mediawiki/2017/e/ef/Ocean_crude_oil_pumpandtreat.png"/>
 
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<h3>EXPERIMENTAL DESIGN</h3>  
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Waste Treatment
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In order to assess whether the newly engineered E. coli strains containing the fluorene catabolic pathway was able to degrade their respective PAH, they were grown in minimal medium supplemented with fluorene as a sole source of carbon. For controls, the strains were grown in presence of glucose. In addition, E. coli strains containing the corresponding vector without insert was also grown in parallel. Fluorene was prepared as stock solution of 10 mg/mL and was initially dissolved in methanol. Because the stock solution showed a slight precipitate, stock solutions of 10 mg/mL was also prepared in the organic solvent dimethyl sulfoxide (DMSO). Growth comparisons using these 2 solvents were performed in parallel.
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Leaking waste containers, oil deposits, and landfills contributing to PAH contamination can be targeted for bioremediation efforts. Centers for remediation efforts may use our bacteria to degrade specific PAH compounds within groundwater in the affected areas.  
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<img src="https://static.igem.org/mediawiki/2017/0/0d/Waste_treatment.png"/>
  
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<h3>CULTURE SETUP</h3>
 
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Cultures were started from glycerol stock in 4 mL of medium and incubated at 37°C. The OD readout of the overnight cultures was determined using a spectrophotometer according to the protocol shown above. All cultures were then diluted to 0.02 using the volume below and OD measurements were determined at the indicated time points.
 
 
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<h3>CONCLUSION</h3>
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Byproduct Management
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The data show all absorbance measurements obtained during the biotransformation of fluorene by our recombinant E. coli in minimal medium supplemented with PAHs. To evaluate whether the recombinant cells had the ability to transform PAHs, growth experiments were set up with various clones expressing the fluorene catabolic pathway. The clones described above with the catabolic pathway under the control of 3 different constitutive promoters were set in cultures using minimal medium supplemented with fluorene (0.1 mg/mL) as sole source of carbon (figures below). Antibiotics were added as appropriately.
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Clones containing the catabolic pathway exhibited higher cell density at 48 hours compared to their respective controls (vector alone) with the strongest promoter given a greater advantage (clone 48 or K2491013 for fluorene). PAHs dissolved in DMSO appeared slightly more available than PAHs dissolved in methanol. This may be explained by the fact that PAHs stock solutions prepared in methanol exhibited some precipitates not observed with DMSO-based stocks.
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Industrial byproducts may be filtered into waste management treatments in order for bioremediation process to target phenanthrene, fluorene, and even naphthalene compounds located in industrial waste products like plastics, pesticides, and oil products.  
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<img src="https://static.igem.org/mediawiki/2017/c/c4/Byproduct_management.png"/>
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<img src="https://2017.igem.org/File:MMFluGrowth2017.png">
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[[Image:MMFluGrowth2017.png|600px]]
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[[Image:MMFluGrowthMeth2017.png|600px]]
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Hazard Identification
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Growth of CCA_San_Diego’s bacteria in PAH contaminated water environments signifies the existence of contaminants in reservoir systems in the absence of a carbon source. Future combination of an identification system as well as our pathway can be used for contamination identification.
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<img src="https://static.igem.org/mediawiki/2017/f/fc/Hazardous_identification.png"/>
 
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Figure 1. Growth of recombinant E. coli BL21DE3 cultures harboring the control plasmid pSB3T5 or the fluorene pathway under the control of 3 different constitutive promoters: BBa_J23100 (clone 48), BBa_J23101 (clone 51), and BBa_J23110 (clone 54) cloned into pSB3T5. Data points represent value averages of the duplicate of OD at 600 nm taken over time for 2 independent colonies per clone. Recombinant clones were grown in minimal medium supplement with tetracycline (15 µg/mL) and fluorene (0.1 mg/mL). Fluorene was dissolved in 100% DMSO (Top panel) or 100% methanol (Bottom panel).
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Reaction Completion Identification
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For future research design, reporter plasmids can be used to identify the intermediates of salicylate and phthalate in order to signify the completion of degradation of PAHs for efficient and quick processing of contaminated water.  
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<img src="https://static.igem.org/mediawiki/2017/e/e6/Reaction_completion_identification.png"/>
 
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Growth comparison of recombinant E. coli in minimal medium supplemented with PAHs and glucose. In order to evaluate the role of toxicity and/or the metabolic burden caused by the PAH catabolic genes and the PAHs, namely fluorene, cells were grown in minimal medium together with fluorene and glucose as carbohydrate sources (figures below). It appears that all 3 clones carried by a low copy plasmid number with the fluorene catabolic pathway under the control of 3 promoters of various strengths behaved similarly to the control strain harboring the corresponding vector pSB3T5 with no insert. The fluorene genes independently of their expression level do not appear to impact cell growth when carried by a low copy vector. In addition, fluorene (0.1 mg/mL) in presence of glucose is not toxic to the cells.  
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<h3>Why hasn’t this been done before?</h3>
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There are several enzymes involved in the PAH upper catabolic pathway. The aerobic degradation of aromatic compounds is typically initiated by oxygenases that catalyze the incorporation of two oxygen atoms into the aromatic ring followed by a dehydrogenation reaction catalyzed by dehydrogenases. Theses enzymes are designated RHDO (ring hydroxylating dioxygenases). The dioxygenases responsible for the first step in the aerobic oxidation of lower molecular weight aromatic hydrocarbons, e.g. naphthalene, biphenyl, benzene, and certain other aromatic compounds, share many similarities with genes for which the sequence is known. In contrast, there is little information about bacterial genes encoding proteins for the degradation of higher molecular weight PAH such as phenanthrene and fluorene.
 
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[[Image:MMGrowthOverTime.png|600px]]
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<h3>What are we doing?</h3>
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To be able to degrade as many aromatic compounds as possible, our approach involves converging the various degradation pathways and augmenting the genes, which is possible through common intermediates. To that end, we would clone the genes upstream of the common intermediates and introduce them into a bacteria that would already contain one or more degradation pathways already containing genes downstream of the intermediates. The host strain already has a piece of the pathway, and by a process of engineering, we are augmenting its gene pool and its capacity to degrade.  
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Figure 3. Fluorene biotransformation experiment using recombinant E. coli BL21DE3 harboring the control plasmid pSB3T5 or the fluorene pathway under the control of 3 different constitutive promoters: BBa_J23100 (clone 48), BBa_J23101 (clone 51), and BBa_J23110 (clone 54) cloned into pSB3T5. Data points represent value averages of duplicate of OD at 600 nm taken over time for 2 independent colonies per clone. Recombinant clones were grown in minimal medium supplement with tetracycline (15 µg/mL), fluorene (0.1 mg/mL), and glucose (0.4%).
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<img src="<img src="https://static.igem.org/mediawiki/2017/e/e6/Reaction_completion_identification.png"/>"/>
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EXPERIMENTAL DESIGN
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In the first phase of our research, a comprehensive literature and genome search was conducted to identify microorganisms with published annotated genes capable of degrading the most abundant PAH in crude oils.
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This study was aimed at determining the growth rate of the recombinant strains containing the fluorene catabolic pathway alone or together when using fluorene as sole source of carbon. Recombinant cells were grown in minimal medium supplemented with fluorene as a sole source of carbon. For controls, the strains were grown in presence of glucose. In addition, E. coli strains containing the corresponding vector without insert was also grown in parallel. PAHs were dissolved in DMSO.
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In the second phase, operons were designed as synthetic genes to exclude restriction enzyme sites reserved for prefix and suffix sequences and to improve the %GC content for optimum expression in E. coli. In addition, RBS sequences were introduced between open reading frames and an inducible promoter was cloned upstream of the operons.
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CULTURE SETUP
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In the third phase, the catabolic pathways were cloned into a RK2 plasmid to have the potential to be introduced into various strains of bacteria for the purpose of gene augmentation.
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<br></br>
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Cultures were started from glycerol stock in 4 mL of medium and placed at 37°C. The OD readout of the overnight cultures was determined using a spectrophotometer according to the protocol shown above. All cultures were then diluted to 0.02 using the volume below and OD measurements were determined at the indicated time points.
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[[Image:MoreGrowthTestFlur.png|600px]]
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Graph 3. Average absorbance values measured in quadruplet at 600 nm of cultures of 2 independent colonies of control (vector) and catabolic plasmid (clone CCA-48 or K2491013 for fluorene) at 2, 4, 24, and 48 hours after inoculation of minimal media supplemented with fluorene at 0.1 mg/mL with or without glucose (0.4%) at 25. All media contained the surfactant Tween-20 (0.1%). Cultures were grown at 25°C or 37°C. MM= Minimal Medium, Glu= Glucose; Ave.= Average; OD=Optical Density; SD= standard deviation.
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<div id="results">
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<h2 id="results" style="/*position:center;*//*position:center;*/text-align:center;position:1000px;padding-top:50px;font-size:79px;color:#56cfff;">Results</h2>
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Here are our results for our project...
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Revision as of 03:01, 2 November 2017

Project Description

Why are PAHs important?

Contamination of aquatic and terrestrial environments with crude oils (or petroleum) represents a fundamental problem worldwide that results in both immediate and long-term environmental damages, including toxicity to plants and soil invertebrates and with significant risks to humans and wildlife. Crude oils are predominantly composed of hydrogen and carbon. The massive ecological damage caused by oil spills in the ocean can largely be attributed to a subset of crude oils called polycyclic aromatic hydrocarbons. Polycyclic aromatic hydrocarbons account for approximately 80% of the total petroleum hydrocarbons in crude oils. Aromatic carbons such as polycyclic aromatic hydrocarbons (PAHs) tend to be the most toxic and persistent molecular compounds in oil.


Applications of PAH-Degrading Bacteria

Contamination of aquatic and terrestrial environments with crude oils (or petroleum) represents a fundamental problem worldwide that results in both immediate and long-term environmental damages, including toxicity to plants and soil invertebrates in addition to significant risks to humans and wildlife.

Applications of PAH-Degrading Bacteria


Isolation and Understanding PAH Degradation Systems
CCA_San_Diego has isolated and modified two major PAH degradation pathways: fluorene and phenanthrene, in order to further understand the scope, efficiency, and application of these pathways for commercial use. These isolated systems serve to limit unwanted bacterial byproducts by allowing the selection of specific strains for expression, to allow for selection of optimal bacteria in field situations, and to reduce the use of harmful bacterial strains that contain many of the pathways we isolated.

Ocean Crude Oil Pump and Treatment
CCA_San_Diego designed bacteria to aid in the process of bioremediation for pump and treat ex-situ methods. Once spills are identified, contaminated sites may be pumped to remediation centers that contain our synthetic PAH-degredetory bacteria. Please see our applied design page for more information about this process.


Waste Treatment
Leaking waste containers, oil deposits, and landfills contributing to PAH contamination can be targeted for bioremediation efforts. Centers for remediation efforts may use our bacteria to degrade specific PAH compounds within groundwater in the affected areas.


Byproduct Management
Industrial byproducts may be filtered into waste management treatments in order for bioremediation process to target phenanthrene, fluorene, and even naphthalene compounds located in industrial waste products like plastics, pesticides, and oil products.


Hazard Identification
Growth of CCA_San_Diego’s bacteria in PAH contaminated water environments signifies the existence of contaminants in reservoir systems in the absence of a carbon source. Future combination of an identification system as well as our pathway can be used for contamination identification.


Reaction Completion Identification
For future research design, reporter plasmids can be used to identify the intermediates of salicylate and phthalate in order to signify the completion of degradation of PAHs for efficient and quick processing of contaminated water.


Why hasn’t this been done before?

There are several enzymes involved in the PAH upper catabolic pathway. The aerobic degradation of aromatic compounds is typically initiated by oxygenases that catalyze the incorporation of two oxygen atoms into the aromatic ring followed by a dehydrogenation reaction catalyzed by dehydrogenases. Theses enzymes are designated RHDO (ring hydroxylating dioxygenases). The dioxygenases responsible for the first step in the aerobic oxidation of lower molecular weight aromatic hydrocarbons, e.g. naphthalene, biphenyl, benzene, and certain other aromatic compounds, share many similarities with genes for which the sequence is known. In contrast, there is little information about bacterial genes encoding proteins for the degradation of higher molecular weight PAH such as phenanthrene and fluorene.

What are we doing?

To be able to degrade as many aromatic compounds as possible, our approach involves converging the various degradation pathways and augmenting the genes, which is possible through common intermediates. To that end, we would clone the genes upstream of the common intermediates and introduce them into a bacteria that would already contain one or more degradation pathways already containing genes downstream of the intermediates. The host strain already has a piece of the pathway, and by a process of engineering, we are augmenting its gene pool and its capacity to degrade.
"/>
In the first phase of our research, a comprehensive literature and genome search was conducted to identify microorganisms with published annotated genes capable of degrading the most abundant PAH in crude oils.
In the second phase, operons were designed as synthetic genes to exclude restriction enzyme sites reserved for prefix and suffix sequences and to improve the %GC content for optimum expression in E. coli. In addition, RBS sequences were introduced between open reading frames and an inducible promoter was cloned upstream of the operons.
In the third phase, the catabolic pathways were cloned into a RK2 plasmid to have the potential to be introduced into various strains of bacteria for the purpose of gene augmentation.

email igemcca@gmail.com
Canyon Crest Academy iGEM 2017 CC;