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== '''Project Overview''' == | == '''Project Overview''' == | ||
- | Haloalkanes are widely used commercially. The | + | Haloalkanes are widely used commercially. The majority 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 pesticide γ-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 can work in ''E. coli'' as well.[1] 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 results showed that all enzymes could co-express as a soluble protein with P2A peptide acting as a linker and F2A could work as the same as in eukaryotic system. Moreover, the resulting engineered ''E. coli'' exhibited an excellent capability for the degradation of TCP and γ-HCH. |
== '''Project Story'''== | == '''Project Story'''== | ||
==='''γ-isomer of hexachlorocyclohexane (γ-HCH)''' === | ==='''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 ] Mounting concerns about its nontarget toxicity and persistence have since caused it to be deregistered in most | + | 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 countries[2], 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] |
- | + | ||
- | + | ||
=== '''1,2,3-Trichloropropane (TCP)''' === | === '''1,2,3-Trichloropropane (TCP)''' === | ||
- | 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 | + | 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 noly contaminate soil, it also 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 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]. |
- | == '''Four Key | + | == '''Four Key Enzymes''' == |
=== LinA and LinB === | === LinA and LinB === | ||
- | Selected LinA and LinB are cofactor-independent dehalogenase from Pseudomonas paucimobilis UT26, LinA mediates | + | 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. |
<html> | <html> | ||
<div align="left" style="width:50%;float:left"> | <div align="left" style="width:50%;float:left"> | ||
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</br> | </br> | ||
</br> | </br> | ||
- | + | Fig.1 Proposed pathway for the metabolism of lindane (-yhexachlorocyclohexane) in ''P. paucimobilis UT26''[3,4,5]. 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; | |
</br> | </br> | ||
</br> | </br> | ||
Line 130: | Line 128: | ||
=== DhaA and HheC === | === DhaA and HheC === | ||
- | 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- | + | 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-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 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] |
- | 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 | + | |
<html> | <html> | ||
<div align="left"> | <div align="left"> | ||
- | + | <img src="https://static.igem.org/mediawiki/igem.org/f/fe/Uestclifeproject3.jpg" /> Fig.2 Degradation pathway of 1,2,3-Trichloropropane (TCP)</a> | |
</div> | </div> | ||
</html> | </html> | ||
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== '''Strategy''' == | == '''Strategy''' == | ||
===2A peptide Sequence === | ===2A peptide Sequence === | ||
- | In foot-and-mouth disease virus (FMDV) and some other picornaviruses the oligopeptide ( | + | In foot-and-mouth disease virus (''FMDV'') and some other picornaviruses, the oligopeptide (≈20 amino acids) 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 years, there was little report about 2A peptide that is active in prokaryotic systems, even some papers stated 2A sequences can’t work in prokaryote. But Indian Scientists Dechamma et al. have found the F2A peptide could work in ''E.coli''[1], which gives us creative advice. |
+ | <html> | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/6/67/Uestclifeproject4.png" /><br/> | ||
+ | Fig.3 the picture comes from http://viralzone.expasy.org/all_by_protein/914.html. | ||
+ | </div> | ||
+ | <p>In our project, at first step we constructed that F2A peptide sequence linked gene ''LinA'' and ''LinB'', and P2A linked gene ''DhaA'' and ''HheC''. (Fig.4) </p> | ||
+ | |||
+ | </html> | ||
+ | |||
+ | :::Fig.4 https://static.igem.org/mediawiki/igem.org/3/33/POHC05.png https://static.igem.org/mediawiki/igem.org/d/d2/POHC06.png | ||
=== Polycistroinc expression === | === Polycistroinc expression === | ||
- | Modular bacterial polycistronic expression system allows | + | Modular bacterial polycistronic expression system allows co-expression and co-purification 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 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. |
+ | <html> | ||
- | <h2 class="acc_trigger"><strong>Reference</strong></h2> | + | </html> |
+ | </br> | ||
+ | :::Fig.5.1 https://static.igem.org/mediawiki/igem.org/3/32/POHC07.jpg https://static.igem.org/mediawiki/igem.org/4/49/POHC08.jpg Fig.5.2 | ||
+ | <h2 class="acc_trigger"><strong>Reference</strong>(click here to see details)</h2> | ||
<div class="acc_container" style="display: none; "> | <div class="acc_container" style="display: none; "> | ||
<!--Content Goes Here--> | <!--Content Goes Here--> | ||
1.H J Dechamma. 2007. Processing of multimer FMD virus VP1-2A protein expressed in E.coli into monomers. Indian Journal of Experimental Biology. | 1.H J Dechamma. 2007. Processing of multimer FMD virus VP1-2A protein expressed in E.coli into monomers. Indian Journal of Experimental Biology. | ||
+ | |||
2.Rup Lal,Gunjan Pandey,Pooja Sharma,Kirti Kumari,Shweta Malhotra,Rinku Pandey.Mar.2010, | 2.Rup Lal,Gunjan Pandey,Pooja Sharma,Kirti Kumari,Shweta Malhotra,Rinku Pandey.Mar.2010, | ||
- | 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 | + | 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 and Prospects for Bioremediation |
- | and Prospects for Bioremediation | + | |
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. | 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. | ||
+ | |||
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. | 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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system. Chemosphere35:1887-1894 | system. Chemosphere35:1887-1894 | ||
- | 8 | + | 8.Lammel, G., Y. S. Ghimb, A. Gradosa, H. Gaod, H. Hu¨hnerfussc, and R.Lohmann.2007. Levels of persistent organic pollutants in air in China and over the Yellow Sea. Atmos. Environ.41:452-464 |
- | Lohmann.2007. Levels of persistent organic pollutants in air in China and | + | |
- | over the Yellow Sea. Atmos. Environ.41:452-464 | + | 9.Li, J., T. Zhu, F. Wang, X. H. Qiu, and W. L. Lin.2006. Observation of organochlorine pesticides in the air of the Mt. Everest region. Ecotoxicol.Environ. Safe.63:33-41 |
- | 9 | + | |
- | organochlorine pesticides in the air of the Mt. Everest region. Ecotoxicol. | + | 10.Kumari, B., V. K. Madan, and T. S. Kathpal.2007. Status of insecticide |
- | Environ. Safe.63:33-41 | + | |
- | 10 | + | |
contamination of soil and water in Haryana, India. Environ. Monit. Assess. | contamination of soil and water in Haryana, India. Environ. Monit. Assess. | ||
136:239-244 | 136:239-244 | ||
- | 11 | + | 11.Prakash, O., M. Suar, V. Raina, C. Dogra, R. Pal, and R. Lal.2004. |
Residues of hexachlorocyclohexane isomers in soil and water samples from | Residues of hexachlorocyclohexane isomers in soil and water samples from | ||
Delhi and adjoining areas. Curr. Sci.87:73-77. | Delhi and adjoining areas. Curr. Sci.87:73-77. | ||
- | 12 | + | 12.Raina, V., M. Suar, A. Singh, O. Prakash, M. Dadhwal, S. K. Gupta, C. |
Dogra, K. Lawlor, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal.2008. | Dogra, K. Lawlor, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal.2008. | ||
Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation withSphingobium indicumB90A. Biodegradation 19:27-40 | Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation withSphingobium indicumB90A. Biodegradation 19:27-40 | ||
- | 13 | + | 13.Toteja, G. S., A. Mukherjee, S. Diwakar, P. Singh, and B. N. Saxena.2003. |
Residues of DDT and HCH pesticides in rice samples from different geographical regions of India: a multicentre study. Food Addit. Contam.20: | Residues of DDT and HCH pesticides in rice samples from different geographical regions of India: a multicentre study. Food Addit. Contam.20: | ||
933-939. | 933-939. | ||
- | 14 | + | 14.Blasco, C., C. M. Lino, Y. Pico, A. Pena, G. Font, and M. I. Silveira.2004. |
Determination of organochlorine pesticide residues in honey from the | Determination of organochlorine pesticide residues in honey from the | ||
central zone of Portugal and the Valencian community of Spain. J. Chromatogr. A1049:155-160 | central zone of Portugal and the Valencian community of Spain. J. Chromatogr. A1049:155-160 | ||
- | 15 | + | 15.Bajpai, A., P. Shukla, B. S. Dixit, and R. Banerji.2007. Concentrations of |
organochlorine insecticides in edible oils from different regions of India. | organochlorine insecticides in edible oils from different regions of India. | ||
Chemosphere67:1403-1407. | Chemosphere67:1403-1407. | ||
- | 16 | + | 16.Bhatnagar, V. K., R. Kashyap, S. S. Zaidi, P. K. Kulkarni, and H. N. |
Saiyed.2004. Levels of DDT, HCH, and HCB residues in human blood in | Saiyed.2004. Levels of DDT, HCH, and HCB residues in human blood in | ||
Ahmedabad, India. Bull. Environ. Contam. Toxicol.72:261-265. | Ahmedabad, India. Bull. Environ. Contam. Toxicol.72:261-265. | ||
- | 17 | + | 17.Subramaniam, K., and R. D. J. Solomon.2006. Organochlorine pesticides |
- | BHC and DDE in human blood in and around Madurai, India. Indian | + | BHC and DDE in human blood in and around Madurai, India. Indian J. Clin. Biochem.21:169-172. |
- | J. Clin. Biochem.21:169-172. | + | |
- | 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. | + | 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. 70099-2240/02/$04.000 DOI: 10.1128/AEM.68.7.3582-3587.2002 |
- | + | ||
19.Cooke, Mary,2009. ?Emerging Contaminant--1,2,3-Trichloropropane (TCP)? (Report). United States EPA. | 19.Cooke, Mary,2009. ?Emerging Contaminant--1,2,3-Trichloropropane (TCP)? (Report). United States EPA. | ||
20.Agency for Toxic Substances and Disease Registry (1992). Toxicological Profile for 1,2,3-Trichloropropane? (Report). U.S. CDC. | 20.Agency for Toxic Substances and Disease Registry (1992). Toxicological Profile for 1,2,3-Trichloropropane? (Report). U.S. CDC. | ||
+ | |||
21. Basic Questions and Answers for the Drinking Water Strategy Contaminant Groups Effort (Report). US EPA. 2011. | 21. Basic Questions and Answers for the Drinking Water Strategy Contaminant Groups Effort (Report). US EPA. 2011. | ||
+ | |||
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. | 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. | ||
Latest revision as of 01:19, 28 September 2013
Project |
---|
Contents |
Project Overview
Haloalkanes are widely used commercially. The majority 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 pesticide γ-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 can work in E. coli as well.[1] 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 results showed that all enzymes could co-express as a soluble protein with P2A peptide acting as a linker and F2A could work as the same as in eukaryotic 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] Mounting concerns about its nontarget toxicity and persistence have since caused it to be deregistered in most countries[2], 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]
1,2,3-Trichloropropane (TCP)
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 noly contaminate soil, it also 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 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].
Four Key Enzymes
LinA and LinB
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.
![](https://static.igem.org/mediawiki/2013/f/fb/Uestclifeproject2.png)
DhaA and HheC
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-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 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]
![](https://static.igem.org/mediawiki/igem.org/f/fe/Uestclifeproject3.jpg)
Strategy
2A peptide Sequence
In foot-and-mouth disease virus (FMDV) and some other picornaviruses, the oligopeptide (≈20 amino acids) 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 years, there was little report about 2A peptide that is active in prokaryotic systems, even some papers stated 2A sequences can’t work in prokaryote. But Indian Scientists Dechamma et al. have found the F2A peptide could work in E.coli[1], which gives us creative advice.
![](https://static.igem.org/mediawiki/2013/6/67/Uestclifeproject4.png)
Fig.3 the picture comes from http://viralzone.expasy.org/all_by_protein/914.html.
In our project, at first step we constructed that F2A peptide sequence linked gene ''LinA'' and ''LinB'', and P2A linked gene ''DhaA'' and ''HheC''. (Fig.4)
- Fig.4
- Fig.4
Polycistroinc expression
Modular bacterial polycistronic expression system allows co-expression and co-purification 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 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. </br>
- Fig.5.1
Fig.5.2
- Fig.5.1
Reference(click here to see details)