Team:Caltech/Project

Bioremediation of Endocrine Disruptors Using Genetically Modified Escherichia Coli

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 17a-ethynylestradiol. 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 created constructs containing previously discovered BisdA and BisdB enzymes that have been shown to degrade BPA and we are testing their efficacy. 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. We were able to establish that our synthesized gene expresses a protein that consistently degrades DDT. This enzyme may also be useful in degrading other EDCs. 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 17a-ethynylestradiol. 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

Plasmid map of BisdA under the lac promoter [http://partsregistry.org/Part:BBa_K620002 K620002] in pSB4A5.

Plamid map of BisdB under the tet promoter [http://partsregistry.org/Part:BBa_K620003 K6230003]

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 listed, 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 are now inducing production of BisdA and BisdB so that we can investigate their ability to degrade EDCs.

Gibson Assembly

A gel showing Gibson assembly of GFP, promoter, terminator and pSB4A5 and BisdA, lac promoter, terminator and pSB4A5 (pNT001) stopped at 0, 5, 10, 15, 30, 60 minutes. Generally, over time, the smaller bands decrease in brightness and larger bands increase in brightness

We attempted to use Gibson Assembly for creating composite BioBrick plasmids including inducible BisdA and BisdB. After many weeks of doing Gibson reactions and screening for colonies, we have observed that Gibson assembly may not be the most efficient and reliable method of assembling BioBricks. Gibson assembly may be a quicker method of cloning than using restriction enzymes and ligase, but this work indicates that quality and quantity of plasmids produced using this method are not as easily reproducible as standard assembly for assembly of multiple BioBrick parts is. If iGEM teams wish to use this method for assembly, we have shown that limiting the total DNA in the Gibson reaction, limiting the number of parts being combined in the reaction and using extremely competent cells improves the ratio of experimental colonies to negative control colonies. These can then be screened using colony PCR or sequencing. However, the high complementarity between the BioBrick prefix and suffix could contribute to the high numbers of self-ligation we observed. Due to the enzymes in the Gibson reaction, phosphatase cannot be used, a common procedure in standard assembly.

We recommend PCR assembly, if possible, of composite BioBrick inserts rather than multi-step standard assembly to increase the efficiency of assembling of BioBrick parts. Future iGEM teams are advised to try different methods of assembly and cloning in parallel, as the parts are not as modular as stated and each combination behaves differently than others.

DDT Dehydrochlorinase

The band indicating DDT dehydrochlorinase is visible in both elution columns, between the 21,500 and 31,000 g/mole bands. The DDT protein is 23,500 g/mole.

GCMS of DDT showing a clear band at 235g/mole

DDT Molecular Structure

DDT structure after GCMS with a molecular weight of 235g/mole

DDT reaction with DDT Dehydrochlorinase analyzed with GCMS. There is a band at 191g/mole and a band at 206 g/mole

DDT structure after reaction with DDT Dehydrochlorinase and GCMS with a molecular weight of 206g/mole

We found DDT Dehydrochlorinase in the literature as an enzyme discovered to degrade DDT. We found the amino acid code for this enzyme on GenBank. We used [http://helixweb.nih.gov/dnaworks/ DNAWorks] to design oligos for assembling this enzyme optimized in E. coli. We assembled this gene using PIPE cloning. We then inserted 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 next conducted degradation experiments with DDT using electrospray GCMS. As shown in the GCMS of DDT without degradation enzymes, there is a clear band at 235 grams/mole. DDT's molar mass is 355. This indicates that the original structure of DDT loses a carbon and three chlorines to form a structure with this mass.

Cytochrome p450s

HPLC-electrospray of BPA

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, 17a-ethinylestradiol, 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 results.

Selection of EDC-Degrading Organisms

Figure 1: Liquid minimal media cultures plated on LB; week 3

Figure 2: Liquid minimal media cultures plated on LB; week 8

In order to identify an organism capable of degrading our EDCs, we collected dry and wet samples from the Los Angeles river and used these samples to inoculate liquid minimal media cultures in which the sole source of carbon was the EDC. (We used media with and without a vitamin mix to account for any compounds the organisms might not be capable of manufacturing themselves, but the presence or absence of the vitamin mix seems to have made little difference in the growth of the organisms; cultures exhibited equivalent growth both with and without the vitamin mix.) We sequentially used these cultures to inoculate new cultures every two to three days over an eight-week period in order to further isolate organisms.

At the third week we plated the cultures on LB plates and observed significant growth of many different types of organisms (figure 1), indicating that our cultures contained organisms capable of surviving on our EDCs. To further isolate these organisms, colonies from each culture were resuspended in liquid minimal media; as before, new liquid minimal media cultures were inoculated from each of these every two to three days. The cultures were plated again on LB in the eighth week (figure 2) and again, significant growth was observed.

At this stage, we attempted to plate our cultures on solid minimal media; however, all of the compounds we chose are insoluble in water, which makes traditional plating impossible. We attempted several alternate methods of plating the EDCs together with the minimal media and cultures, but all these attempts were unsuccessful. Thus, although we demonstrated the presence of EDC-degrading organisms in our cultures, we were unable to fully isolate any organisms for further study.

Biofilm Columns

Visible layers of biofilm as visualized by crystal violet