Progress 04/01/09 to 03/31/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We reported on this National Institute of Environmental Health and Science award as a state project simply so that it would appear on our financial report template. Closing the project in REEport now that it has ended.
Publications
|
Progress 04/01/12 to 03/31/13
Outputs
OUTPUTS: 1. Chlorpyrifos removal and degradation by wild-type plants. Ms. Keum Young Lee has been working on her doctoral dissertation in Professor Doty's laboratory focusing on using plants for taking up and breaking down the common agricultural pesticide, chlorpyrifos. She tested a variety of poplar and willow plants. 2. Engineering Transgenic Poplar Overexpressing PON1 for Improved Degradation of chlorpyrifos. Our previous work showed that we successfully transformed hybrid poplar with mammalian PON1 (paraoxonase 1), which is known to be involved in chlorpyrifos (CPS) metabolism in mammals. There was no significant difference between wild type and transgenic plants for phytotoxicity, CPS removal, and CPS degradation. To investigate whether the pon1 gene was making active PON1 protein in transgenic plants, a his-tag was added. The his-tagged PON1 was inserted in a plant expression vector (pART27) and used to transform E. coli and Agrobacterium. 3. PON1 Foam Technology via Chloroplast Genetic Engineering. Our previous work showed that a tobacco clone was been successfully transformed with PON1 via chloroplast genetic engineering, through which DNA in chloroplasts was modified, instead of the nuclear DNA. However, there was no functional expression in the PON1 transgenic tobacco, for CPS phytotoxicity, CPS uptake, and PON1 enzyme activity assay. A his-tagged PON1 construct was inserted into a tobacco chloroplast expression vector (pLD2-CtV) and expressed in bacteria by E. coli transformation. In addition, to see if there is transient PON1 gene expression in plants, GFP (green fluorescent protein) was added to the tobacco chloroplast expression vector. PARTICIPANTS: Stuart Strand, PI. Research Professor, Department of Civil and Environmental Engineering and School of Forest Resources, University of Washington. Sharon Doty, Co-PI. Associate Professor, School of Environmental and Forest Sciences, University of Washington. Keum Young Lee, Graduate Research Assistant, School of Environmental and Forest Sciences, University of Washington. TARGET AUDIENCES: Target audiences include bioremediation scientists, civil and environmental engineers, governmental agencies, nongovernmental organizations, land managers, educators, students, and the general public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. Chlorpyrifos removal and degradation by wild-type plants. Ms. Keum Young Lee discovered that many varieties of poplar and willow plants can successfully remove chlorpyrifos from the environment and naturally degrade it. She is currently doing time course experiments with willow to determine the chlorpyrifos degradation pathway. 2. Engineering Transgenic Poplar Overexpressing PON1 for Improved Degradation of chlorpyrifos. The his-tagged PON1 protein was not detected by western blot. Apparently there was no PON1 protein in standard E. coli strains or in plants. Previously, functional PON1 enzyme was only achieved using a specialized E. coli strain. 3. PON1 Foam Technology via Chloroplast Genetic Engineering. The his-tagged PON1 protein was not detected. It seems fairly clear that there was no PON1 protein produced in E. coli. The engineered vector was first expressed in E. coli, but there was no fluorescence generated. A new construct was prepared to more strongly express foreign proteins. When the RBS (ribosome binding site) of the vector was replaced from "ggagg" to 'phage T7 gene 10 leader (g10L)' sequence, there was very strong green fluorescence, suggesting that the g10L enhances the expression of GFP. In order to investigate whether g10L RBS enhances the expression of PON1, g10L sequence will be added to the tobacco chloroplast expression vector containing PON1 instead of the normal RBS sequence, and PON1-GFP expression will be tested. Similar constructs but without the GFP fusion are being prepared as well to test for increased PON1 enzyme activity in vitro.
Publications
- Lee, K. Y., Strand, S. E., and Doty, S. L. 2012. Phytoremediation of chlorpyrifos by Populus and Salix. International Journal of Phytoremediation 14(1):48-61.
|
Progress 04/01/11 to 03/31/12
Outputs
OUTPUTS: (1) Field-scale demonstration of 2E1 transgenic poplar ability to take up TCE: PROGRESS: A controlled field experiment to compare the abilities of transgenic poplar expressing the mammalian cytochrome P450 2E1 (CP4502E1) to phytoremediate groundwater contaminated with trichloroethylene (TCE) under field conditions is ongoing. Planted and control beds are dosed with TCE, and effluent samples are analyzed. During the last season we also sampled soil chloroethenes. (2) Phytoremediation of Chlorpyrifos using CYP & PON1 Transgenic Poplars: PROGRESS: Seven poplar and willow varieties were screened for the ability to take up chlorpyrifos from contaminated water. Our results were published this year in the International Journal of Phytoremediation. (3) PON1 Foam Technology via Chloroplast Genetic Engineering: PROGRESS: We continued efforts to improve the remediation capacity of poplar using transgenic technologies. The mammalian genes, PON1 and CYPB6 for chlorpyrifos degradation were inserted in the nuclear genome of poplar and the chloroplast genome of tobacco and these transgenes were effectively transcribed. The graduate student on the project learned a great deal about plant tissue culture, two methods of plant transformation, molecular biology, and biochemistry. She presented her work at the International Phytotechnologies meeting this September and won first place for best poster presentation. (4) Increase the uptake of VOCs from the air by transgenic plants: PROGRESS: With the help of two talented undergraduate volunteers we have resumed efforts to engineer CYP4502E1 into a houseplant, pothos ivy and to determine whether increased stomatal opening increases uptake of VOCs. PARTICIPANTS: Stuart Strand, PI. Research Professor, Department of Civil and Environmental Engineering and School of Forest Resources, University of Washington. Sharon Doty, Co-PI. Associate Professor, School of Forest Resources, University of Washington. Indulis Muiznieks, Research Technologist, Department of Civil and Environmental Engineering, University of Washington. Kimberly Young, Lab Manager/Research Engineer, Department of Civil and Environmental Engineering, University of Washington. Keith Stewart, Graduate Research Assistant, Department of Civil and Environmental Engineering, University of Washington. Keum Young Lee, Graduate Research Assistant, School of Forest Resources, University of Washington. TARGET AUDIENCES: Target audiences include bioremediation scientists, civil and environmental engineers, governmental agencies, nongovernmental organizations, land managers, educators, students, and the general public. PROJECT MODIFICATIONS: Note that budgetary reductions have led to dropping the following Specific Aims: (1) Determine the ecotoxicity of root materials, leaf litter and rhizosphere soil of CYP2E1 transgenic plants exposed to VOCs. (2) Clone and analyze the plant genes involved in the degradation of TCE and its metabolites, and other pollutants.
Impacts
(1) Field-scale demonstration of 2E1 transgenic poplar ability to take up TCE: RESULTS: We found that significantly more TCE was lost in the planted beds than in the unplanted beds, although loss of water by transpiration in the planted beds was high enough that effluent TCE concentrations were higher than in the unplanted beds. Preliminary data showed that the TCE recovery from the transgenic tree beds was lower than the wild type tree beds, indicating that transgenic trees increased the removal of TCE in under field conditions. Dehalococcoides 16S ribosomal genes were detected by nested PCR but associated functional genes tceA, vcrA, and bvcA, which are responsible for chlorinated ethene degradation, were not found. qPCR analysis of DNA samples from each bed revealed the presence of aerobic the functional genes etnC and etnE, associated with the aerobic oxidation of vinyl chloride. Together these data suggest that TCE was removed by reductive dechlorination of trichloroethene to cis-1,2-dichloroethene and vinyl chloride, and aerobic oxidation to ethene. PLAN: We are finishing an analysis of chloroethenes in the soils of the field beds to obtain a better chloroethenes accounting. We will quantify the presence of tceA, vcrA, bvcA genes, the 16S ribosomal genes of Dehalococcoides, and etnC and etnE functional genes using qPCR. (2) Phytoremediation of Chlorpyrifos using CYP & PON1 Transgenic Poplars: RESULTS: Although all seven poplar and willow varieties were able to remove the pollutant, there were clear differences in uptake rate, with specific willow lines removing the pesticide faster. Not only did the plants take up the pollutant, they also translocated it throughout the plant and degraded it nearly completely by an unknown mechanism over several weeks. PLAN: We are currently conducting studies to determine where the system is blocked, either protein production or active enzyme formation. (3) PON1 Foam Technology via Chloroplast Genetic Engineering: RESULTS: So far our efforts have not been successful. No active enzyme has been detected. Furthermore, the transgenic plants do not have increased removal of the pesticide from solution or any reduction in phytotoxic effects. PLAN: We are currently conducting studies to determine where the system is blocked, either protein production or active enzyme formation. (4) Increase the uptake of VOCs from the air by transgenic plants: RESULTS: Transformed pothos is presently regenerating from callus tissue cultures. Arabidopsis mutants with increased stomata have been transformed with the CYP4502E1 gene and are presently being screened. PLAN : CYP4502E1 transformed polthos will be tested for uptake and degradation of chloroform and benzene, common household air pollutants. CYP4502E1 transformed Arabidopsis stomatal overexpressing mutants will be tested for chloroform uptake compared to wild type.
Publications
- Lee, K. Y., Strand, S. E., and Doty, S. L. 2012. Phytoremediation of Chlorpyrifos by Populus and Salix, Intern. Jour. Phytoremediation. 14 (1) 48-61.
|
Progress 04/01/10 to 03/31/11
Outputs
OUTPUTS: 1) Field study of transgenic poplars for TCE phytoremediation: A controlled field experiment to compare the abilities of transgenic poplar expressing the mammalian cytochrome P450 2E1 (CP4502E1) to phytoremediate groundwater contaminated with trichloroethylene (TCE) under field conditions is ongoing. The University of Washington Phytoremediation Field Site consists of lined beds containing no plants (control), wild type poplar, and transgenic poplars expressing (CP4502E1). These beds are dosed with TCE contaminated water and the effluent concentrations of TCE are measured by GC-ECD analysis in the laboratory. 2) Phytoremediation of Chlorpyrifos using CYP & PON1 Transgenic Poplars: Transgenic expression of genes involved in CPS metabolism is expected to increase removal of CPS. Cytochrome P450s (CYP) activate CPS in mammals, forming CPS-oxon, which paraoxonase 1 (PON1) detoxifies. Human CYP2B6 and recombinant rabbit PON1 have been cloned into separate plant expression vectors and used to simultaneously transform a poplar hybrid clone using the Agrobacterium-mediated method. 3) PON1 Foam Technology via Chloroplast Genetic Engineering: In order to express the PON1 protein at high levels for inexpensive, large-scale production of this remediation tool, we proposed to use the chloroplast genome transformation method. To this end, rabbit PON1 was cloned into the chloroplast transformation vector and the vector DNA were bombarded onto tobacco leaf tissue using a particle delivery system. In November 2010, Ms. Keum Young Lee attended the annual Superfund meeting in Portland, OR. She presented a poster entitled "Three Approaches to Enhancing Phytoremediation of Chlorpyrifos" at the poster session. (Awarded 2nd place as the "Best Student Poster"). PARTICIPANTS: Stuart Strand, PI. Research Professor, Department of Civil and Environmental Engineering and School of Forest Resources, University of Washington. Sharon Doty, Co-PI. Associate Professor, School of Forest Resources, University of Washington. Indulis Muiznieks, Research Technologist, Department of Civil and Environmental Engineering, University of Washington. Kimberly Young, Lab Manager/Research Engineer, Department of Civil and Environmental Engineering, University of Washington. Keith Stewart, Graduate Research Assistant, Department of Civil and Environmental Engineering, University of Washington. Keum Young Lee, Graduate Research Assistant, School of Forest Resources, University of Washington. TARGET AUDIENCES: Target audiences include bioremediation scientists, civil and environmental engineers, governmental agencies, nongovernmental organizations, land managers, educators, students, and the general public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Field study of transgenic poplars for TCE phytoremediation: Although the transgenic trees processed more TCE-contaminated water and had more TCE metabolites in the tissues, overall they did not reduce the concentration of TCE in the water or have reduced phytotranspiration of TCE. 2) Phytoremediation of Chlorpyrifos using CYP & PON1 Transgenic Poplars: We have confirmed the presence of CYP2B6 and PON1 in putative transgenic lines using polymerase chain reaction (PCR). We have obtained 10 transgenic lines expressing both genes (rabbit PON1 and CYP2B6) and 35 transgenic plants expressing only rabbit PON1. Total RNA was isolated from confirmed transgenic leaf tissues and the level of transgene expression was analyzed by RT (Reverse Transcriptase)-PCR. Transgenic poplar lines showing high level of expression were selected for further study. These are now being propagated in preparation for biochemistry experiments. 3) PON1 Foam Technology via Chloroplast Genetic Engineering: We have obtained spectinomycin-resistant, PON1 putative transformed tobacco plants, and confirmed the transgene integration in putative transplastomic plants by PCR. Total RNA was isolated from confirmed transgenic leaf tissues and the level of transgene expression was analyzed by RT-PCR. This chloroplast transgenic tobacco shows much higher expression compared to nuclear transgenic poplar. Propagation of transgenic tobacco by tissue culture is in progress for further study, and some tobacco plants are being grown for seed collection.
Publications
- Kang, J. W., Wilkerson, H.-W., Farin, F. M., Bammler, T. K., Beyer, R. P., Strand, S. E. and Doty, S. L. 2010. Mammalian cytochrome CYP2E1 triggered differential gene regulation in response to trichloroethylene (TCE) in a transgenic poplar. Funct Integr Genomics. 2010 Aug;10(3):417-24.
|
Progress 04/01/09 to 03/31/10
Outputs
OUTPUTS: 1. Field-Scale Demonstration of 2E1 Transgenic Poplar Ability to Take Up Trichloroethylene (TCE): A controlled field experiment to compare the abilities of transgenic poplar expressing the mammalian cytochrome P450 2E1 (CP4502E1) to phytoremediate groundwater contaminated with TCE under field conditions is ongoing. Our field site consists of lined beds containing no plants (control), wild type poplars, and transgenic poplars. These beds are dosed with TCE contaminated water and the effluent concentrations of TCE are measured. 2. Isolation and Characterization of TCE-Inducible Plant Genes: Tricholoethanol (TCOH) is conjugated to sugars in tobacco plants exposed to TCE or TCOH and upon hydrolosis with beta-glucosidase, a much higher level of TCOH is released compared to the free TCOH in the control. In previous work we found that glucosyltransferases (GTF) expression was unregulated in poplar exposed to TCE and TCOH. To characterize the GTF identified by array analysis, we expressed the GTFs in a recombinant yeast expression system. 3. Phytoremediation of Chlorpyrifos: Our previous work showed that chlorpyrifos can be taken up and accumulated by hydroponic plant of poplar and willow. The best performing willow line was treated with chlorpyrifos. We will continue our work: 1. Monitoring and comparing effluent water from beds containing hybrid poplars and wild type poplars. Further research will include stable isotope (SI) fractionation to determine the fractionation produced by plant uptake of TCE and application of SI fractionation to determine the relative contribution of rhizospheric bacteria in TCE breakdown. We will also apply genetic markers of reductive and oxidative microbial metabolism of TCE and its breakdown products to the microbial population of the test bed rhizospheres. 2. Assessing whether aquaporins transport TCE, by working with radiolabeled glycerol/trichloroethelene uptake in mutant Arabidopsis plants. 3. Our best performing willow line will be treated with chlorphyrifos, and the uptake and degradation will be monitored over a time course. In order to increase the chlorpyrifos phytoremediation potential of plants, we are overexpressing the enzymes required for its degradation: Cytochrome P450 2B6 activates chlorpyrifos in mammals, forming chlorpyrifos-oxon, which paraoxonase 1 (PON1) detoxifies. Human cytochrome P450 2B6 and recombinant rabbit PON1 have been cloned into separate plant expression vectors and were used to simultaneously transform our poplar hybrid clone. Putative transgenic plants are currently growing; the presence of the two transgenes will be confirmed. We will then analyze the plants for increased uptake and degradation of chlorpyrifos. In order to express the PON1 protein at high levels for inexpensive, large-scale production of this remediation tool, we proposed to use the chloroplast genome transformation method. We cloned the PON1 complementary DNA (cDNA) into the appropriate vector, and have begun particle bombardment of tobacco leaf tissue experiments. The transgenic plants will be screened for maximum production of PON1 protein for use in the foam technology. PARTICIPANTS: Stuart Strand, PI. Research Professor, Department of Civil and Environmental Engineering and School of Forest Resources, University of Washington. Sharon Doty, Co-PI. Associate Professor, School of Forest Resources, University of Washington. Indulis Muiznieks, Research Technologist, Department of Civil and Environmental Engineering, University of Washington. Kimberly Young, Lab Manager/Research Engineer, Department of Civil and Environmental Engineering, University of Washington. Keith Stewart, Graduate Research Assistant, Department of Civil and Environmental Engineering, University of Washington. Keum Young Lee, Graduate Research Assistant, School of Forest Resources, University of Washington. TARGET AUDIENCES: Target audiences include bioremediation scientists, civil and environmental engineers, governmental agencies, nongovernmental organizations, land managers, educators, students, and the general public. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1. Field-scale demonstration of 2E1 transgenic poplar ability to take up trichloroethylene (TCE): Unexpectedly the effluent TCE concentrations were highest for the transgenic trees, followed by the wild type, and the control bed. These results suggest that microbial activity (e.g., reductive dechlorination) dominates TCE metabolism in poplar phytoremediation environments and/or that plant uptake and metabolism of TCE is limited by transport of TCE into roots. Current experiments to determine whether TCE uptake occurs through aquaporins, would suggest additional genetic modifications that would increase TCE phytoremediation. Planning of microbial community analysis is also underway. 2. Isolation and Characterization of TCE-Inducible Plant Genes: The selected glucosyltransferases (GTF) has been successfully cloned into a yeast expression vector. Yeast containing the vector will be treated with trichloroethanol (TCOH), harvested, and analyzed for free and glycosylated TCOH. 3. Phytoremediation of Chlorpyrifos: Chlorpyrifos did not persist in the plants, suggesting metabolism of chlorpyrifos in plant tissue. Cytochrome P450s activate chlorpyrifos in mammals, forming chlorpyrifos-oxon, which paraoxonase 1 (PON1) detoxifies. Human cytochrome P450 2B6 and recombinant rabbit PON1 have been cloned into separate plant expression vectors and used to simultaneously transform our poplar hybrid clone. The presence of the two transgenes will be confirmed using polymerase chain reaction (PCR) and investigated for uptake and degradation of chlorpyrifos. In order to express the PON1 protein at high levels for inexpensive, large-scale production of this remediation tool, we proposed to use the chloroplast genome transformation method. To this end, we requested the appropriate plasmid from Professor Henry Daniell (University of Central Florida) who invented this technique. We germinated surface-sterilized tobacco seeds at the same time (July 2009) and have grown the plants in sterile tissue culture. The plasmid and protocol were sent to us (October 2009), and we are now in the process of cloning the PON1 cDNA into this vector in preparation for particle bombardment of tobacco leaf tissue.
Publications
- James, C.A., Xin, G., Doty, S.L., Muiznieks, I., Newman, L.A., and Strand, S.E. 2009. A Mass Balance Study of the Phytoremediation of Perchloroethylene-Contaminated Groundwater. Environmental Pollution. 157 (8-9) 2564-2569.
- James, C.A., and Strand, S.E. 2009. Phytoremediation of Small Organic Contaminants Using Transgenic Plants. Current Opinion in Biotechnology. 20 (2) 237-241.
|
|