Difference between revisions of "Team:UFAM-UEA Brazil/Project/Phytochelatin"

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<p id="t2">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam lorem elit, molestie in nunc id, viverra iaculis augue. Praesent sed gravida lorem, vel sollicitudin turpis. Mauris efficitur ligula blandit lorem auctor lacinia. Etiam vitae justo nibh. Curabitur vehicula purus auctor lacus vestibulum feugiat. Pellentesque ex dui, pretium nec odio vitae, efficitur volutpat risus. Curabitur mollis tortor et hendrerit sodales. Nam id leo sit amet libero varius luctus. Nullam mollis mauris nec magna iaculis cursus. Nunc convallis fringilla nisl, ut scelerisque urna laoreet in. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed vitae ante viverra, luctus dolor et, rutrum tellus. Etiam in mollis turpis. Ut eu tellus id nunc congue cursus. Phasellus non leo ut ante ultricies sodales. Mauris congue lectus dui. </p>
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<p id="t2"><p>In the environment Hg has successive transformations  which pose risks not only for microorganisms but also to macro fauna. However its known that some bacteria specie has mercury resistance, among them Serratia marcescens, Pseudomonas putida, Cupriavidus metallidurans and Entereobacter. Bacterial resistance to mercury occurs due to  membrane protein expression that can act in Hg capture. Among those we can find the phytochelatin. These proteins have as main feature the interaction with heavy metals. Probably this occurs due to the great amount of cystein amino acid in this protein. </p>
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<p>The use of natural membrane proteins is already in place and serve as a  tool to anchor heterologous proteins in a system called “cell surface display”. It presents a great potential for a variety of biotech uses. By this strategy target peptides could be anchored to antibodies production, biocatalizers, bioremediation and other uses. In heavy metal bioremediation  its is showed that recombinant microorganisms with modified surface, enriched with metal chelant proteins are better to cope the adsorption  of  metallic ions. </p>
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<p>There are several strategies to anchor peptides in the bacterial membrane. In this project we used the most abundant protein to do so, the E. coli  outer membrane protein A (OmpA) – fused with synthetic phytochelatin to bioremediation of mercury metal. In 2000 a new series of peptides serving as heavy metal adsorbants was proposed by Bae and collaborators. The strategy consisted in the use of an analogous to a natural phytochelatin without the necessity  of post-transdutional modifications to work without  using enzymatic routes or precursor molecules to its, in other words: a gene a protein. In previous competition we tested the efficiency of a Metal Binding Protein (https://2014.igem.org/Team:UFAM_Brazil/Bioaccumulation ). In this project we design a system for Hg bioaccumulation by a synthetic phytochelatin anchored in the membrane protein  OmpA of a host bacteria. </p>
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<p>REFERENCES</p>
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</br>
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<p>1.    BIONDO, R. Engenharia Genética de Cupriavidusmetallidurans para a biorremediação de efluentes contendo metais pesados, 2008. </p>
 +
</br>
 +
 
 +
<p>2.   BAE, W.; MEHRA, R.K. Metal-binding characteristics of a phytochelatins analog (GluCys)2Gly.J. Inorg. Biochem., v. 68, p. 201-210, 1997. </p>
 +
</br>
 +
 
 +
<p>3.    BAE, W.; CHEN, W.; MULCHANDANI, A.; MEHRA R. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol. Bioeng., v. 70, p. 518-524, 2000. </p>
 +
</br>
 +
 
 +
<p>4.    BAE, W.; MEHRA, R.K.; MULCHANDANI, A.; CHEN, W. Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury. Appl. Environ. Microbiol., v. 67, n. 11, p. 5335-5338, 2001. </p>
 +
</br>
 +
 
 +
<p>5.    COSTA, G. S. Aplicação De Biossensor Microbiano Bioluminescente Na Detecção De HG (II), 2010. </p>
 +
</br>
 +
 
 +
<p>6.    GIOVANELLA, P.; BENTO, F.; CABRAL, L.; GIANELLO, C.; CAMARGO, F. A. O.Isolamento e seleção de microrganismos resistentes e capazes de volatilizar mercúrio, 2010. </p>
 +
</br>
 +
 
 +
<p>7.    NASCIMENTO, A. M. A.; CHARTONE-SOUZA. E. Operon mer: Bacterial resistance to mercury and potential for bioremediation of contaminated environments, 2003. </p>
 +
</br>
 +
 
 +
<p>8.    NEVES-PINTO, M. Bases moleculares da resistência ao mercúrio em bactérias gram-negativas da Amazônia brasileira, 2004. </p>
 +
</br>
 +
 
 +
<p>9.    SAMBROOK, J.; RUSSEL, D.W. Molecular Cloning: a Laboratory Manual. 3rd ed. Cold Spring HarborLaboratory Press, New York 2001. </p>
 +
</br>
 +
 
 +
<p>10. SHEILA, S. S. Estudo do gene merA em bactérias gram-negativas resistentes ao mercúrio isoladas de ecossistemas aquáticos brasileiros: contribuição para a mitigação dos riscos do mercúrio à saúde humana através da biorremediação, 2012. </p>
 +
</br>
 +
 
 +
<p>11. SOUZA, J. R.; BARBOSA, A. C. Contaminação por Mercúrio e o caso da Amazônia, 2000. </p>
 +
</br>
 +
 
 +
<p>12. GREEN, M.R. e SAMBROOK, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, 4ª Ed. Cold Spring Harbour, USA, 2012. </p>
 +
</br>
 +
 
 +
<p>13. SHETTY, R. P.; ENDY, D.; KNIGHT, T. F. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. Cambridge, MA, USA, 2008. </p>
 +
</br>
 +
 
 +
<p>14. DASH, H. R.; DAS, S. Bioremediation of mercury and the importance of bacterial mer genes International Biodeterioration & Biodegradation. Orissa, India, 2012. </p>
 +
 
 +
</br>
 +
 
 +
<p>15. WASSERMAN, J. C.; HACON, S. S.; WASSERMAN, M. A. O Ciclo do Mercúrio no Ambiente Amazônico. Mundo & Vida Vol. 2. Niterói, RJ, Brasil, 2001. </p>
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 +
</br>
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 +
</p>
 
 
  

Revision as of 19:24, 19 October 2016

Phytochelatin

Description

In the environment Hg has successive transformations which pose risks not only for microorganisms but also to macro fauna. However its known that some bacteria specie has mercury resistance, among them Serratia marcescens, Pseudomonas putida, Cupriavidus metallidurans and Entereobacter. Bacterial resistance to mercury occurs due to membrane protein expression that can act in Hg capture. Among those we can find the phytochelatin. These proteins have as main feature the interaction with heavy metals. Probably this occurs due to the great amount of cystein amino acid in this protein.



The use of natural membrane proteins is already in place and serve as a tool to anchor heterologous proteins in a system called “cell surface display”. It presents a great potential for a variety of biotech uses. By this strategy target peptides could be anchored to antibodies production, biocatalizers, bioremediation and other uses. In heavy metal bioremediation its is showed that recombinant microorganisms with modified surface, enriched with metal chelant proteins are better to cope the adsorption of metallic ions.



There are several strategies to anchor peptides in the bacterial membrane. In this project we used the most abundant protein to do so, the E. coli outer membrane protein A (OmpA) – fused with synthetic phytochelatin to bioremediation of mercury metal. In 2000 a new series of peptides serving as heavy metal adsorbants was proposed by Bae and collaborators. The strategy consisted in the use of an analogous to a natural phytochelatin without the necessity of post-transdutional modifications to work without using enzymatic routes or precursor molecules to its, in other words: a gene a protein. In previous competition we tested the efficiency of a Metal Binding Protein (https://2014.igem.org/Team:UFAM_Brazil/Bioaccumulation ). In this project we design a system for Hg bioaccumulation by a synthetic phytochelatin anchored in the membrane protein OmpA of a host bacteria.



REFERENCES


1. BIONDO, R. Engenharia Genética de Cupriavidusmetallidurans para a biorremediação de efluentes contendo metais pesados, 2008.


2. BAE, W.; MEHRA, R.K. Metal-binding characteristics of a phytochelatins analog (GluCys)2Gly.J. Inorg. Biochem., v. 68, p. 201-210, 1997.


3. BAE, W.; CHEN, W.; MULCHANDANI, A.; MEHRA R. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol. Bioeng., v. 70, p. 518-524, 2000.


4. BAE, W.; MEHRA, R.K.; MULCHANDANI, A.; CHEN, W. Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury. Appl. Environ. Microbiol., v. 67, n. 11, p. 5335-5338, 2001.


5. COSTA, G. S. Aplicação De Biossensor Microbiano Bioluminescente Na Detecção De HG (II), 2010.


6. GIOVANELLA, P.; BENTO, F.; CABRAL, L.; GIANELLO, C.; CAMARGO, F. A. O.Isolamento e seleção de microrganismos resistentes e capazes de volatilizar mercúrio, 2010.


7. NASCIMENTO, A. M. A.; CHARTONE-SOUZA. E. Operon mer: Bacterial resistance to mercury and potential for bioremediation of contaminated environments, 2003.


8. NEVES-PINTO, M. Bases moleculares da resistência ao mercúrio em bactérias gram-negativas da Amazônia brasileira, 2004.


9. SAMBROOK, J.; RUSSEL, D.W. Molecular Cloning: a Laboratory Manual. 3rd ed. Cold Spring HarborLaboratory Press, New York 2001.


10. SHEILA, S. S. Estudo do gene merA em bactérias gram-negativas resistentes ao mercúrio isoladas de ecossistemas aquáticos brasileiros: contribuição para a mitigação dos riscos do mercúrio à saúde humana através da biorremediação, 2012.


11. SOUZA, J. R.; BARBOSA, A. C. Contaminação por Mercúrio e o caso da Amazônia, 2000.


12. GREEN, M.R. e SAMBROOK, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, 4ª Ed. Cold Spring Harbour, USA, 2012.


13. SHETTY, R. P.; ENDY, D.; KNIGHT, T. F. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. Cambridge, MA, USA, 2008.


14. DASH, H. R.; DAS, S. Bioremediation of mercury and the importance of bacterial mer genes International Biodeterioration & Biodegradation. Orissa, India, 2012.


15. WASSERMAN, J. C.; HACON, S. S.; WASSERMAN, M. A. O Ciclo do Mercúrio no Ambiente Amazônico. Mundo & Vida Vol. 2. Niterói, RJ, Brasil, 2001.


Results

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam lorem elit, molestie in nunc id, viverra iaculis augue. Praesent sed gravida lorem, vel sollicitudin turpis. Mauris efficitur ligula blandit lorem auctor lacinia. Etiam vitae justo nibh. Curabitur vehicula purus auctor lacus vestibulum feugiat. Pellentesque ex dui, pretium nec odio vitae, efficitur volutpat risus. Curabitur mollis tortor et hendrerit sodales. Nam id leo sit amet libero varius luctus. Nullam mollis mauris nec magna iaculis cursus. Nunc convallis fringilla nisl, ut scelerisque urna laoreet in. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed vitae ante viverra, luctus dolor et, rutrum tellus. Etiam in mollis turpis. Ut eu tellus id nunc congue cursus. Phasellus non leo ut ante ultricies sodales. Mauris congue lectus dui.

Notebook

1st Day


-Preparation of electrocompetente cells of JM110 and DH5α.

-Parts transformation (JK26 promoter, Lpp-OmpA BBa_K103006, Phytochelatin and Terminator BBa_B0015).


2nd Day

-Isolation in plate (four transforming per plate)


3rd Day


-Inoculum of four transforming colonies from the parts (JK26 promoter, Lpp-OmpA BBa_K103006, Phytochelatin and Terminator BBa_B0015) isolated the previous day.


4th Day


-Plasmidial extraction of the inoculum with kit Illustra plasmidPrep Mini Spin GE Healthcare.

Gel extraction of parts in agarose gel 0,8%; 1, 2, 3, 4) P1 promoter; 5, 6, 7, 8) Lpp-OmpA (OmpA); 9, 10, 11, 12) Phytochelatin (PTC); 13, 14, 15, 16) Terminator BBa_B0015; Colonies 1, 2, 3, 4 of each part respectively.

-Test digestion to confirm the sizes with EcoRI

Digestion test of the parts in agarose gel 0,8%; 1, 3, 5, 7) P1 Promoter – Not Digested; 2, 4, 6, 8) P1 Promoter – Digestion with EcoRI.; 9, 11, 13, 15) Omp A – Not Digested.; 10, 12, 14, 16) Omp A – Digestion with EcoRI; 17, 19, 21, 23) FTQ – Not Digested.; 18, 20, 22, 24) FTQ – Digestion with EcoRI.; 25, 27, 29, 31) Terminator – Not Digested.; 26, 28, 30, 32) Terminator – Digestion with EcoRI.; Colonies 1, 2, 3, 4 of each part respectively.

-Digestion to confirm inserts.

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aliquam lorem elit, molestie in nunc id, viverra iaculis augue. Praesent sed gravida lorem, vel sollicitudin turpis. Mauris efficitur ligula blandit lorem auctor lacinia. Etiam vitae justo nibh. Curabitur vehicula purus auctor lacus vestibulum feugiat. Pellentesque ex dui, pretium nec odio vitae, efficitur volutpat risus. Curabitur mollis tortor et hendrerit sodales. Nam id leo sit amet libero varius luctus. Nullam mollis mauris nec magna iaculis cursus. Nunc convallis fringilla nisl, ut scelerisque urna laoreet in. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed vitae ante viverra, luctus dolor et, rutrum tellus. Etiam in mollis turpis. Ut eu tellus id nunc congue cursus. Phasellus non leo ut ante ultricies sodales. Mauris congue lectus dui.