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| - | == '''Project Overview''' == | + | == '''TCP Biodegradation achieved''' == |
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| - | Haloalkanes are widely used commercially. The majorty of these compounds have been shown to be serious pollutants as they are toxic and quite persistent in the environment, such as a man-made industrial chemical 1,2,3-Trichloropropane (TCP) and an organic pesticideγ-Hexachlorocyclohex-ane (Lindane，γ-HCH). These halogenated compounds have been introduced into our environment as a consequence of industrial waste disposal and widespread open use in agriculture, which need to be removed to low levels from waste streams and during sanitation of polluted sites. For this reason, Microbial degradation of these compounds represents an important and efficient way to fulfill the target. In order to improve biodegradation efficiency, several powerful genetically engineered E. coli strains have been constructed by the co-expression of key enzymes involving in the biodegradation pathways of the two compounds. As putting the different selected enzymes together, it will have an ability to degrade more halogenated compounds besides the both. To construct the efficient co-expression system and achieve biodegradation of γ-HCH and TCP, these key enzymes, LinA, LinB, DhaA and HheC, are chosen, and foot and mouth disease virus 2A peptide and polycistronic co-expression strategies were adopted. Foot and mouth disease virus 2A peptide has been widely used for co-expression of multiple genes in eukaryote systems. However, the use of the 2A peptide in prokaryotes is limited, and so far only one paper described that F2A ca in E. coli as well.1To explore whether the 2A peptides can work in our co-expression system, several vectors were constructed by using all three 2A peptides, respectively. The results showed that all enzymes could co-expressed as a soluble protein with P2A peptide acting as a linker and F2A could function the same as in eukaryote system. Moreover, the resulting engineered E. coli exhibited an excellent capability for the degradation of TCP and γ-HCH.
| + | Growth of E.coli strain MC1061 transformed DhaA+P2A+HheC and DhaA gene in LB medium with inducer and 5mM TCP respectively, as the substrate was monitored in batch culture. The concentration of the products were confirmed using GC column(AC5). Growth resulted in disappearance of the substrate and simultaneous formation of biomass, 2,3-DCP, epichlorohydrin, chloropropanol, and glycerin. The result was screened by GC analysis. Above the degradation of TCP and the intermediate product 2,3-DCP indicate that multistep biodegradation are working. (Fig.1 and Fig.2). And cultivating in medium with 2,3-DCP, the concentration of epichlorohydrin can be detected(Fig.2). It means HheC degrade 2,3-DCP to epichlorohydrin. The degradation followed the desired path, TCP will be degraded to glycerol finally. The deficiency of our project is chloropropanol and glycerin couldn’t be detected by the columns. In the future, new columns and new methods will be used. |
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| - | == '''Project Story'''==
| + | <center>https://static.igem.org/mediawiki/2013/4/40/Uestcliferesult2.jpg</center> |
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| - | ==='''γ-isomer of hexachlorocyclohexane (γ-HCH)''' ===
| + | Fig.1. E.coli MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and it mediate product 2,3-DCP were measured by GC. 2,3-DCP was degraded at the same time. |
| - | HCH, formerly known as benzene hexachloride (BHC), is one of the now notorious organochlorine group of insecticides. About 600,000 tons were used throughout the world between the 1940s and the 1990s to control a wide range of agricultural, horticultural, and public health pests.[2 3 ] Mounting concerns about its nontarget toxicity and persistence have since caused it to be deregistered in most countries2, but it is very stable in the environment[2 7] and it is still being manufactured in India for local and export uses, so residue problems will continue for many decades.2 The majority of HCH waste has been discarded in the open or stored at various levels of containment near the production sites, HCH residues at many of the sites have percolated into the soil and then contaminated ground water.2 Residues of HCH have now been reported for many countries in samples of air[8 9] water[10] soil[10 11 12 ]food commodities[13 14 15 ] and even from human blood samples.[16 17]
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| | + | Fig.2. E.coli MC1061 carried DhaA incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP couldn’t be degraded. |
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| - | === '''1,2,3-Trichloropropane (TCP)''' ===
| + | <center>https://static.igem.org/mediawiki/2013/f/fa/Uestcliferesult4.jpg</center> |
| | + | Fig.3. E.coli MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with 2,3-DCP(5mM) at 30℃. The concentration of 2,3-DCP and its mediate product epichlorohydrin were measured by GC. |
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| - | 1,2,3-Trichloropropane (TCP) is a toxic synthetic chlorinated hydrocarbon that is not known to occur naturally[20]. It is used as a chemical intermediate in organic synthesis, as a solvent, and as an extractive agent. In addition to these intentional uses, TCP is produced in considerable amounts as a by-product from the manufacture of epichlorohydrin. TCP does not contaminate soil. Instead, it leaks down into groundwater and settles down at the bottom of the ground water reservoir because TCP is more dense than water. This makes TCP in its pure form a DNAPL (Dense Nonaqueous Phase Liquid) and it is therefore harder to remove it from groundwater[19]. Groundwater and soils in various parts of the United States and in Europe are polluted with TCP as a result of improper disposal of TCP-contaminated effluents and due to the past use of the soil fumigant D-D, a mixture of 1,3-dichloropropene and 1,2-dichloropropane that contained TCP as a contaminant (U.S. Environmental Protection Agency, national priority site fact sheet and Tysons dump site). Due to its toxicity and persistence, TCP poses a serious risk to ecosystems and human health[18 19 21].
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| - | == '''Four Key Enzyme''' ==
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| - | === LinA and LinB ===
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| - | Selected LinA and LinB are cofactor-independent dehalogenase from Pseudomonas paucimobilis UT26, LinA mediates the first step of aerobic microbial degradation of γ-HCH to 1,3,4,6-tetrachloro-1,4-cyclohexadiene , which is further metabolized by the sequential activity of LinB.
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| - | FIG.1 Proposed pathway for the metabolism of lindane (-yhexachlorocyclohexane) in P. paucimobilis UT26 (352). 1,-y-Hexachlorocyclohexane; 2, y-pentachlorocyclohexene; 3, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (chemically unstable); 4, 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (chemically unstable); 5, 2,5dichloro-2,5-cyclohexadiene-1,4-diol;
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| - | === DhaA and HheC ===
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| - | DhaA and HheC also are cofactor-free dehalohygenase. DhaA from Rhodococcus sp hydrolyzes carbon-halogen bonds in a wide range of haloalkanes, including TCP, to the corresponding haloalcohol, releasing halide ions. Haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 is a potentially useful enzyme involved in the degradation of several important environmental pollutants, such as 1,3-dichloro-2-propanol, 2,3-dichloropropa-
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| - | nol, 1-chloropropanol, epichlorohydrin and so on. Additionally, HheC has highly enantioselective dehalogenation of vicinal haloalcohols to epoxides, as well as the reverse reaction, the enantioselective and â-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. In the synthesis of enantioselective medicine HheC being a important instrument puts it into a high gear. For the efficient degradation, we select two mutants DhaA31 and HheC/W239P, which have higher activity.18,22
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| - | == '''Strategy''' ==
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| - | ===2A peptide Sequence ===
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| - | In foot-and-mouth disease virus (FMDV) and some other picornaviruses the oligopeptide (~20 amino acid) 2A region of the polyprotein mediates "cleavage" at its own C-terminus to release it from the 2B region. 2A is also active when placed between reporter proteins and in all eukaryotic systems tested - it acts as an autonomous element, making it an important tool for co-ordinated synthesis of multiple proteins from one open reading frame and making a ribosome jumping. (FIG.3)In recent year, there is little report about 2A peptide sequence that is active in prokaryotic systems, even some paper stated 2A sequence can’t work in prokaryotic. But Indian Scientist Dechamma et.1 have found the T2A peptide can work in E.coli, which give us creative advice.
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| - | === Polycistroinc expression ===
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| - | Modular bacterial polycistronic expression system allows coexpression and copurification of multiple polypeptides in E. coli from a single expression plasmid. The system is comprised of the polycistronic expression vector and a transfer vector which facilitates subcloning of component genes into the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. In this project, 2A peptide sequence working in E.coli is just a exploration without guaranty from sufficient evident. Polycistroinc expression is the next way to achieve multi-enzyme co-expression.
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| - | <h2 class="acc_trigger"><strong>Reference</strong></h2>
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| - | 1.H J Dechamma. 2007. Processing of multimer FMD virus VP1-2A protein expressed in E.coli into monomers. Indian Journal of Experimental Biology.
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| - | 2.Rup Lal,Gunjan Pandey,Pooja Sharma,Kirti Kumari,Shweta Malhotra,Rinku Pandey.Mar.2010,
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| - | Biochemistry of Microbial Degradation of HexachlorocyclohexaneMICROBIOLOGY AND MOLECULARBIOLOGY REVIEWS p. 58-80 Vol. 74, No. 11092-2172/10/$12.00 doi:10.1128/MMBR.00029-09
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| - | and Prospects for Bioremediation
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| - | 3. Li, Y. F.1999. Global technical hexachlorocyclohexane usage and its contamination consequences in the environment: from 1948 to 1997. Sci. Total Environ.232:121-158.
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| - | 4.Vijgen, J., L. F. Yi, M. Forter, R. Lal, and R. Weber.2006. The legacy of lindane and technical HCH production. Organohalog. Comp.68:899-904.
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| - | 5.Ware, G. W. (ed.).1989. The pesticide book. Thomson Publications,Fresno, CA.
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| - | 6.Weber, R., C. Gaus, M. Tysklind, P. Johnston, M. Forter, H. Hollert, H.Heinisch, I.Holoubek, M. Lloyd-Smith, S. Masunaga, P. Moccarelli, D.Santillo, N. Seike, R. Symons, J. P. M. Torres, M. Verta, G. Varbelow, J.Vijgen, A. Watson, P. Costner, J. Woelz, P. Wycisk, and M. Zennegg.2008.Dioxin- and POP-contaminated sites-contemporary and future relevance
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| - | and challenges. Environ. Sci. Pollut. Res.15:363-393.
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| - | 7.Wu, W. Z., Y. Xu, K.-W. Schramm, and A. Kettrup.1997. Study of sorption,
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| - | biodegradation and isomerization of HCH in stimulated sediment/water
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| - | system. Chemosphere35:1887-1894
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| - | 8.76.Lammel, G., Y. S. Ghimb, A. Gradosa, H. Gaod, H. Hu¨hnerfussc, and R.
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| - | Lohmann.2007. Levels of persistent organic pollutants in air in China and
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| - | over the Yellow Sea. Atmos. Environ.41:452-464
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| - | 9.79.Li, J., T. Zhu, F. Wang, X. H. Qiu, and W. L. Lin.2006. Observation of
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| - | organochlorine pesticides in the air of the Mt. Everest region. Ecotoxicol.
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| - | Environ. Safe.63:33-41
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| - | 10.71.Kumari, B., V. K. Madan, and T. S. Kathpal.2007. Status of insecticide
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| - | contamination of soil and water in Haryana, India. Environ. Monit. Assess.
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| - | 136:239-244
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| - | 11.139.Prakash, O., M. Suar, V. Raina, C. Dogra, R. Pal, and R. Lal.2004.
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| - | Residues of hexachlorocyclohexane isomers in soil and water samples from
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| - | Delhi and adjoining areas. Curr. Sci.87:73-77.
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| - | 12.144.Raina, V., M. Suar, A. Singh, O. Prakash, M. Dadhwal, S. K. Gupta, C.
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| - | Dogra, K. Lawlor, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal.2008.
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| - | Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation withSphingobium indicumB90A. Biodegradation 19:27-40
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| - | 13.175.Toteja, G. S., A. Mukherjee, S. Diwakar, P. Singh, and B. N. Saxena.2003.
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| - | Residues of DDT and HCH pesticides in rice samples from different geographical regions of India: a multicentre study. Food Addit. Contam.20:
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| - | 933-939.
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| - | 14.17.Blasco, C., C. M. Lino, Y. Pico, A. Pena, G. Font, and M. I. Silveira.2004.
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| - | Determination of organochlorine pesticide residues in honey from the
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| - | central zone of Portugal and the Valencian community of Spain. J. Chromatogr. A1049:155-160
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| - | 15.7.Bajpai, A., P. Shukla, B. S. Dixit, and R. Banerji.2007. Concentrations of
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| - | organochlorine insecticides in edible oils from different regions of India.
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| - | Chemosphere67:1403-1407.
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| - | 16.15.Bhatnagar, V. K., R. Kashyap, S. S. Zaidi, P. K. Kulkarni, and H. N.
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| - | Saiyed.2004. Levels of DDT, HCH, and HCB residues in human blood in
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| - | Ahmedabad, India. Bull. Environ. Contam. Toxicol.72:261-265.
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| - | 17.169.Subramaniam, K., and R. D. J. Solomon.2006. Organochlorine pesticides
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| - | BHC and DDE in human blood in and around Madurai, India. Indian
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| - | 18.Tjibbe Bosma,Jir Damborsky,Gerhard Stucki,and Dick B. Janssen.2002.Biodegradation of 1,2,3-Trichloropropane through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene APPLIED ANDENVIRONMENTALMICROBIOLOGY,p. 3582-3587 Vol. 68, No. 7
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| - | 0099-2240/02/$04.000 DOI: 10.1128/AEM.68.7.3582-3587.2002
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| - | 19.Cooke, Mary,2009. ?Emerging Contaminant--1,2,3-Trichloropropane (TCP)? (Report). United States EPA.
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| - | 20.Agency for Toxic Substances and Disease Registry (1992). Toxicological Profile for 1,2,3-Trichloropropane? (Report). U.S. CDC.
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| - | 21. Basic Questions and Answers for the Drinking Water Strategy Contaminant Groups Effort (Report). US EPA. 2011.
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| - | 22.Tang L, van Merode AE, Lutje Spelberg JH, Fraaije MW, Janssen DB.2003.Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter. Roles of W139 and W249 in the active site and halide-induced conformational change.Biochemistry.42(47):14057-65.
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Growth of E.coli strain MC1061 transformed DhaA+P2A+HheC and DhaA gene in LB medium with inducer and 5mM TCP respectively, as the substrate was monitored in batch culture. The concentration of the products were confirmed using GC column(AC5). Growth resulted in disappearance of the substrate and simultaneous formation of biomass, 2,3-DCP, epichlorohydrin, chloropropanol, and glycerin. The result was screened by GC analysis. Above the degradation of TCP and the intermediate product 2,3-DCP indicate that multistep biodegradation are working. (Fig.1 and Fig.2). And cultivating in medium with 2,3-DCP, the concentration of epichlorohydrin can be detected(Fig.2). It means HheC degrade 2,3-DCP to epichlorohydrin. The degradation followed the desired path, TCP will be degraded to glycerol finally. The deficiency of our project is chloropropanol and glycerin couldn’t be detected by the columns. In the future, new columns and new methods will be used.
Fig.1. E.coli MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and it mediate product 2,3-DCP were measured by GC. 2,3-DCP was degraded at the same time.
Fig.2. E.coli MC1061 carried DhaA incubated in LB medium with TCP(5mM) at 30℃. The concentration of TCP and its mediate product 2,3-DCP were measured by GC. 2,3-DCP couldn’t be degraded.
Fig.3. E.coli MC1061 carried DhaA+P2A+HheC gene incubated in LB medium with 2,3-DCP(5mM) at 30℃. The concentration of 2,3-DCP and its mediate product epichlorohydrin were measured by GC.
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