Team:UFAM-UEA Brazil/Project/Phytochelatin
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,as represented in the image below. 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.
Phytochelatin model.
In previous competition we tested the efficiency of Metal Binding Peptide. In this project we design a system for Hg bioaccumulation by a synthetic phytochelatin anchored in the membrane protein OmpA of a host bacteria.
One of the pillar of our project is to design the sequences for our genetic modified systems and for that the most challenging step is to adjust preferential codons for our chassis. Glutamic acid (E) and cystein (C) show two different codons in E. coli: GAG and GAA for E; TGC and TGT for C. GAA 70% e códon GAG 30%; códon TGC 60% e códon TGT 40%.
Our synthetic phytochelatin (EC20 - Glu-Cis) optimized to E. coli has: I) 10 cystein amino acids being 12 codons TGC and 8 codons TGT; II) 10 glutamic acid amino acids, 14 codons GAA and 6 codons GAG. As described in the image below.
Codon composition of our Phytochelatin.
With the phytochelatin designed we decided to express it in membrane cell display. For that we incorporated the followed parts:
Promotor JK26 interacting with sigma factor RpoS or sigma factor S to express in the stationary phase in the cell growth. JK26 is a promotor for late phase (lag) described by Miksch et al in 2005 and has been the strongest one fwe tested.
Important to say in this construction, the promotor JK26 with NdeI site was calculated to - when bond to the Llp-OmpA-phytochelatin + terminator – have 9 base pairs in distance between Shine-Dalgarno and the mRNA initiation.
Lpp-OmpA (BBa_K103006) E. coli membrane protein is where our phytochelatin was bond;
The double transcription terminator is the union of T1 from E. coli and TE from T7, denominated as BBa_B0015, available in the Registry.
In silico from Software Snapgene:
To construct our final bioaccumulation device, we followed the steps below:
1st Step:
Phytochelatin digestion with EcoRI + SpeI releasing Lpp-OmpA with NdeI and XbaI. To linearize Lpp-OmpA (2526 bp) and release the promoter (~83 bp).
3rd Step:
Digestion of first step construction, resulting the PO construction (JK26 promoter + Lpp-OmpA) digested with EcoRI + SpeI, releasing a 564 bp insert. The PT (Phytochelatin + Terminator) construction was linearized with EcoRI + XbaI, releasing insert of approximately 2317 bp.
Finally we had our bioaccumulator construction!!
If you want to know how it went in the lab acsses our Phtochelatin notebook.
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
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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 of each 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.
Digestion to confirm insert in agarose gel 1,5%; 1) P1 Promoter – Not Digested.; 2) P1 Promoter – Digested with EcoRI and PstI.; 3) Omp A – Not Digested.; 4) Omp A – Digested with EcoRI and PstI.; 5) PTC – Not Digested.; 6)PTC- Digested with EcoRI and PstI.; 7) Terminator – Not Digested.; 8) Terminator – Digested with EcoRI and PstI.;
5th Day
Analytical digestion for parts purifying.
Extraction gel from the parts in agarose gel 0,8%; 1,2,3,4,5,6,7,8,9) PO – JK26 Promoter linked to Lpp-OMPA.
Purification of parts.
Ligation; promoter to Lpp-OMPA and Phytochelatin to Terminator.
6th Day
Transformation of the ligations in JM110.
7th Day
Isolation in plate of all transforming cells in each linking.
8th Day
Inoculum of transforming isolated from PO (Promoter JK26 and Lpp-OMPA) and PT (Phytochelatin and Terminator) linkings.
9th Day
Plasmidial extraction of the inoculum with Kit Illustra plasmidPrep Mini Spin GE Healthcare.
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Extraction gel from parts in agarose gel 0,8%; 1,2,3,4,5) PT - Phytochelatin linked to Terminator.
Digestion test to confirm the size with EcoRI and PstI.
Digestion test to confirm insert of PO (Promoter and Lpp-OmpA) linking in agarose gel 0,8%;
Digestion to confirm the insert from the PT (Phytochelatin and Terminator) linking in agarose gel 0,8%.
10th Day
Analytical digestion from parts purification
Analytical gel to parts purification in agarose gel 0,8%; 1) PO – Not Digested.; 2) PO – Digested with NotI.; 3) PO – Digested with EcoRI and SpeI.; 4)PT – Not Digested.; 5) PT- Digestion with NotI.; 6) PT – Digestion with EcoRI and XbaI.
Purification.
1) PO – Purified; digested with EcoRI and SpeI; 2)PT – Purified; digestion with EcoRI and XbaI.;
Linking
11th Day
Transformation of POFT linking in DH5α
12th Day
Isolating all POFT linking transforming in plate.
13th Day
Inoculum of isolated transforming from POFT ligation.
14th Day
Plasmidial extraction of inoculum from POFT linking with Kit Illustra plasmidPrep Mini Spin GE Healthcare.
Extraction gel POFT ligation in agarose gel 0,8%.
Digestion test to confirm size with EcoRI and PstI.
Digestion to confirm the insert of POFT linking (Promoter+Lpp-OmpA+Phytochelatin and Terminator) in agarose gel 0,8%.
With the ending of the construction we continued with characterization
