Team:UESTC Life/Project

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Revision as of 01:32, 15 September 2013

Project Overview

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.

Project Story

γ-isomer of hexachlorocyclohexane (γ-HCH)

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 5】 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】

Part 2

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-Hexachlorocyclohexane (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, the biodegradation of halogenated compounds is of great importance. In the project, several key enzymes in the biodegradation pathways of the two compounds were co-expressed in E. coli. As putting the different selected enzymes together, it will have a ability to degrade more halogenated compounds besides the both. To construct the efficient co-expression system and achieve biodegradation of γ-HCH and TCP, we adopt 2A peptide and polycistronic co-expression. 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 can work in E. coli as well. To explore whether the 2A peptides can work in our co-expression system, several vectors were constructed by using all three 2A peptides, respectively.

The Experiments

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-Hexachlorocyclohexane (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, the biodegradation of halogenated compounds is of great importance. In the project, several key enzymes in the biodegradation pathways of the two compounds were co-expressed in E. coli. As putting the different selected enzymes together, it will have a ability to degrade more halogenated compounds besides the both. To construct the efficient co-expression system and achieve biodegradation of γ-HCH and TCP, we adopt 2A peptide and polycistronic co-expression. 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 can work in E. coli as well. To explore whether the 2A peptides can work in our co-expression system, several vectors were constructed by using all three 2A peptides, respectively.

Part 3

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-Hexachlorocyclohexane (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, the biodegradation of halogenated compounds is of great importance. In the project, several key enzymes in the biodegradation pathways of the two compounds were co-expressed in E. coli. As putting the different selected enzymes together, it will have a ability to degrade more halogenated compounds besides the both. To construct the efficient co-expression system and achieve biodegradation of γ-HCH and TCP, we adopt 2A peptide and polycistronic co-expression. 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 can work in E. coli as well. To explore whether the 2A peptides can work in our co-expression system, several vectors were constructed by using all three 2A peptides, respectively.

Results