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Bioremediation of Endocrine Disruptors Using Genetically Modified Escherichia Coli

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Endocrine disruptors, or substances that mimic estrogen in the body, have detrimental biological effects on the reproduction of several species of fish and birds; the Caltech team focuses on bioremediation of these toxins. Our goal is to create a system housed in E. coli that can be used to process water and remove endocrine disruptors on a large scale. We focus on isolating degradation systems for the common endocrine disruptors bisphenol A (BPA), DDT, nonylphenol and estradiol. We synthesized known degradation enzymes DDT dehydrochlorinase, BisdA and BisdB, and characterized the behavior of these enzymes when acting on our target endocrine disruptors. In addition, we explored the potential of certain cytochrome p450s to initiate degradation of these chemicals, focusing on WT-F87A degradation of BPA. Finally, we characterized the functionality of E. coli protein processing when E. coli is deployed as an easily containable biofilm on various substances in aqueous environments.

Introduction:

Endocrine-disrupting chemicals (EDCs) are chemicals that interact with the endocrine system by binding to hormone receptors, causing problems in sexual development and reproduction of organisms. These chemicals are introduced to the environment from improper disposal of plastic wastes, hormonal medications remaining in human waste, and pesticides. Areas with high concentrations of estrogen in water have been shown to correlate with a higher percentage of intersex fish, and pesticides such as DDT have been shown to impact the development of the female reproductive tract in birds. Many EDCs are persistent organic pollutants, and even though regulations have been put in place for pesticide use and industrial production of endocrine disruptors, many of these chemicals continue to pollute bodies of water in significant concentrations.

Synthetic biology involves the engineering of genetic networks to create modified organisms that can address a problem or perform a task. We focus on modifying the genetic code of E. coli to remove endocrine-disrupting chemicals from water. To do this, we investigated previously discovered BisdA and BisdB enzymes that have been shown to degrade BPA and tested their efficacy after production using E. coli. We also assembled a gene for DDT Dehydrochlorinase, an enzyme previously shown to degrade DDT, and expressed this gene in E. coli to characterize its properties. In addition to testing enzymes known to degrade EDCs, we also conducted a search for cytochrome p450s, enzymes known to initiate degradation of many compounds through an oxidation-reduction process. Using p450s selected from the Arnold lab library, we conducted reactions with BPA, DDT, nonylphenol, and estradiol. We analyzed the products of these reactions with HPLC. We found that BPA showed evidence of degradation when combined with the p450 WT-F87A, so the gene for this p450 could be useful for bioremediation.

To further explore the possibilities for bioremediation of EDCs, we performed a selection experiment on biological samples from the Los Angeles River. Since the Los Angeles River is highly polluted with plastics and located in an urban area, it is likely that some selection has already occurred to allow organisms to consume EDCs. We grew these samples on minimal media with various EDCs as carbon sources, and demonstrated the presence of live organisms after several weeks of serial selection, indicating that these organisms can degrade EDCs as a primary carbon source. In order to test the feasibility of an E. coli-housed system for bioremediation, we conducted experiments with biofilms prepared on glass beads in a column. As a model system, we used the degradation of X-gal with beta-galactosidase. We also conducted an evaluation of local water plants to determine typical purification systems for large bodies of water and see if a bioremediation unit can integrate into typical water treatment plants.

BisdA and BisdB

We sourced genetic material for BisdA and BisdB from the Registry of Standard Biological Parts. The sequence listed for these parts had the wrong codons, so we sequenced the parts to determine the correct codons. We then designed genetic constructs with inducible promoters, ribosome binding regions, and terminators in plasmid backbones for each gene. We accessed these parts by transforming DNA from the Registry of Standard Biological Parts into chemically competent E. coli for construction, and used PCR to extract our desired components. We attempted several methods of assembly for these pieces, including traditional assembly, Gibson assembly, and a combination of PCR assembly for the coding construct and standard assembly to insert the coding construct into a backbone. After assembly of these components was complete, we combined the two coding constructs into one vector and expressed this vector in E. coli for experimentation. We then induced production of BisdA and BisdB, and lysed the cells. We mixed this lysate with BPA, DDT, ethinyl estradiol, and nonylphenol, and analyzed the products of this reaction with electrospray high-performance liquid chromatography.

DDT Dehydrochlorinase

We found DDT Dehydrochlorinase in the literature as an enzyme discovered to degrade DDT. We found the amino acid code for this enzyme and assembled a gene codon optimized to produce it in E. coli. We then assembled this gene in a pET vector with a his-tag and overexpressed it in E. coli. We purified the protein and ran it on a gel, indicating that this gene can be expressed in E. coli. We also analyzed this enzyme’s degradation of DDT, estradiol, nonylphenol, and BPA using electrospray HPLC.

Cytochrome p450s

Cytochrome p450s are known to be initiators of degradation for several compounds. We analyzed the structures of the cytochrome p450s which the Arnold Lab possessed in their genetic library and selected four highly promiscuous p450s. We then conducted reactions of these p450s with BPA, DDT, ethinyl estradiol, and nonylphenol. We attempted to analyze the product of these reactions with HPLC and with GCMS, but we found that the product did not stay ionized and therefore could not be analyzed. We formed an electrospray of the reaction products and analyzed this using HPLC, and we were able to see degradation of BPA into a different compound in the HPLC results.

Selection of EDC-Degrading Organisms

We collected dry and wet samples from the LA river and used these samples to inoculate liquid minimal media cultures, with and without a vitamin mix. We then sequentially used these cultures to inoculate new cultures every two to three days over an eight-week period. At the close of this period, we plated the cultures on LB plates and observed significant growth of many different types of organisms. We also conducted a genomic analysis of these samples using 16s sequencing to determine the types of organisms contained in these environmental samples.

Biofilm Columns

To prepare biofilms, we first tested the length of time it takes for a biofilm to form in a 96 well plate using crystal violet.

@@ Line 7: / Line 7: @@
 Endocrine disruptors, or substances that mimic estrogen in the body, have detrimental biological effects on the reproduction of several species of fish and birds; the Caltech team focuses on bioremediation of these toxins. Our goal is to create a system housed in ''E. coli'' that can be used to process water and remove endocrine disruptors on a large scale. We focus on isolating degradation systems for the common endocrine disruptors bisphenol A (BPA), DDT, nonylphenol and estradiol.  We synthesized known degradation enzymes DDT dehydrochlorinase, BisdA and BisdB, and characterized the behavior of these enzymes when acting on our target endocrine disruptors. In addition, we explored the potential of certain cytochrome p450s to initiate degradation of these chemicals, focusing on WT-F87A degradation of BPA. Finally, we characterized the functionality of ''E. coli'' protein processing when ''E. coli'' is deployed as an easily containable biofilm on various substances in aqueous environments.
 <!--- The Mission, Experiments --->
-== '''Overall project''' ==
 ===Introduction:===
 Endocrine-disrupting chemicals (EDCs) are chemicals that interact with the endocrine system by binding to hormone receptors, causing problems in sexual development and reproduction of organisms. These chemicals are introduced to the environment from improper disposal of plastic wastes, hormonal medications remaining in human waste, and pesticides. Areas with high concentrations of estrogen in water have been shown to correlate with a higher percentage of intersex fish, and pesticides such as DDT have been shown to impact the development of the female reproductive tract in birds. Many EDCs are persistent organic pollutants, and even though regulations have been put in place for pesticide use and industrial production of endocrine disruptors, many of these chemicals continue to pollute bodies of water in significant concentrations.<br/><br/>
-Synthetic biology involves the engineering of genetic networks to create modified organisms that can address a problem or perform a task. We focus on modifying the genetic code of E. coli to remove endocrine-disrupting chemicals from water. To do this, we investigated previously discovered BisdA and BisdB enzymes that have been shown to degrade BPA and tested their efficacy after production using E. coli. We also assembled a gene for DDT Dehydrochlorinase, an enzyme previously shown to degrade DDT, and expressed this gene in E. coli to characterize its properties.
+Synthetic biology involves the engineering of genetic networks to create modified organisms that can address a problem or perform a task. We focus on modifying the genetic code of ''E. coli'' to remove endocrine-disrupting chemicals from water. To do this, we investigated previously discovered BisdA and BisdB enzymes that have been shown to degrade BPA and tested their efficacy after production using E. coli. We also assembled a gene for DDT Dehydrochlorinase, an enzyme previously shown to degrade DDT, and expressed this gene in'' E. coli'' to characterize its properties.
 In addition to testing enzymes known to degrade EDCs, we also conducted a search for cytochrome p450s, enzymes known to initiate degradation of many compounds through an oxidation-reduction process. Using p450s selected from the Arnold lab library, we conducted reactions with BPA, DDT, nonylphenol, and estradiol. We analyzed the products of these reactions with HPLC.  We found that BPA showed evidence of degradation when combined with the p450 WT-F87A, so the gene for this p450 could be useful for bioremediation.<br/><br/>
 To further explore the possibilities for bioremediation of EDCs, we performed a selection experiment on biological samples from the Los Angeles River. Since the Los Angeles River is highly polluted with plastics and located in an urban area, it is likely that some selection has already occurred to allow organisms to consume EDCs. We grew these samples on minimal media with various EDCs as carbon sources, and demonstrated the presence of live organisms after several weeks of serial selection, indicating that these organisms can degrade EDCs as a primary carbon source.
-In order to test the feasibility of an E. coli-housed system for bioremediation, we conducted experiments with biofilms prepared on glass beads in a column. As a model system, we used the degradation of X-gal with beta-galactosidase. We also conducted an evaluation of local water plants to determine typical purification systems for large bodies of water and see if a bioremediation unit can integrate into typical water treatment plants.<br/><br/>
+In order to test the feasibility of an'' E. coli''-housed system for bioremediation, we conducted experiments with biofilms prepared on glass beads in a column. As a model system, we used the degradation of X-gal with beta-galactosidase. We also conducted an evaluation of local water plants to determine typical purification systems for large bodies of water and see if a bioremediation unit can integrate into typical water treatment plants.<br/><br/>
 ===BisdA and BisdB===
-We sourced genetic material for BisdA and BisdB from the Registry of Standard Biological Parts. The sequence listed for these parts had the wrong codons, so we sequenced the parts to determine the correct codons. We then designed genetic constructs with inducible promoters, ribosome binding regions, and terminators in plasmid backbones for each gene. We accessed these parts by transforming DNA from the Registry of Standard Biological Parts into chemically competent E. coli for construction, and used PCR to extract our desired components. We attempted several methods of assembly for these pieces, including traditional assembly, Gibson assembly, and a combination of PCR assembly for the coding construct and standard assembly to insert the coding construct into a backbone. After assembly of these components was complete, we combined the two coding constructs into one vector and expressed this vector in E. coli for experimentation. We then induced production of BisdA and BisdB, and lysed the cells. We mixed this lysate with BPA, DDT, ethinyl estradiol, and nonylphenol, and analyzed the products of this reaction with electrospray high-performance liquid chromatography. <br/><br/>
+We sourced genetic material for BisdA and BisdB from the Registry of Standard Biological Parts. The sequence listed for these parts had the wrong codons, so we sequenced the parts to determine the correct codons. We then designed genetic constructs with inducible promoters, ribosome binding regions, and terminators in plasmid backbones for each gene. We accessed these parts by transforming DNA from the Registry of Standard Biological Parts into chemically competent ''E. coli'' for construction, and used PCR to extract our desired components. We attempted several methods of assembly for these pieces, including traditional assembly, Gibson assembly, and a combination of PCR assembly for the coding construct and standard assembly to insert the coding construct into a backbone. After assembly of these components was complete, we combined the two coding constructs into one vector and expressed this vector in ''E. coli'' for experimentation. We then induced production of BisdA and BisdB, and lysed the cells. We mixed this lysate with BPA, DDT, ethinyl estradiol, and nonylphenol, and analyzed the products of this reaction with electrospray high-performance liquid chromatography. <br/><br/>
 ===DDT Dehydrochlorinase===
-We found DDT Dehydrochlorinase in the literature as an enzyme discovered to degrade DDT. We found the amino acid code for this enzyme and assembled a gene codon optimized to produce it in E. coli. We then assembled this gene in a pET vector with a his-tag and overexpressed it E. coli. We purified the protein and ran it on a gel, indicating that this gene can be expressed in E. coli. We also analyzed this enzyme’s degradation of DDT, estradiol, nonylphenol, and BPA using electrospray HPLC.<br/><br/>
+We found DDT Dehydrochlorinase in the literature as an enzyme discovered to degrade DDT. We found the amino acid code for this enzyme and assembled a gene codon optimized to produce it in ''E. coli''. We then assembled this gene in a pET vector with a his-tag and overexpressed it in ''E. coli''. We purified the protein and ran it on a gel, indicating that this gene can be expressed in ''E. coli''. We also analyzed this enzyme’s degradation of DDT, estradiol, nonylphenol, and BPA using electrospray HPLC.<br/><br/>
 ===Cytochrome p450s===
 Cytochrome p450s are known to be initiators of degradation for several compounds. We analyzed the structures of the cytochrome p450s which the Arnold Lab possessed in their genetic library and selected four highly promiscuous p450s. We then conducted reactions of these p450s with BPA, DDT, ethinyl estradiol, and nonylphenol. We attempted to analyze the product of these reactions with HPLC and with GCMS, but we found that the product did not stay ionized and therefore could not be analyzed. We formed an electrospray of the reaction products and analyzed this using HPLC, and we were able to see degradation of BPA into a different compound in the HPLC results.<br/><br/>