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 Study

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

To determine our construction resistance in DH5-alpha transformed with BBa_K2123302. To make plates, the transforming clone with the BBa_K2123302 construction was selected and pre-inoculated alongside a random sample as negative control with its respective antibiotics. After the growth, the optical density was analyzed intending to standardize the density with the control.

Solid LM medium (LB with low concentration of NaCl) used in the plates without adding antibiotic. The pre-inoculated samples were plated and the filter paper disks were put to further add HgCl2 solution at 2000ppm.

The samples were incubated at 37ºC for 16 hours.

The graph represented on Figure 1 shows that our construction with phytochelatin can resist 41% more than our control!!!(is 41% more resistant than our control). As shown in Figure 2 the measurement from the center of the paper filter shows that our construction is capable of getting nearest to the disc than our control which supports our Bioaccumulation device.

With a better notion to which levels of mercury our cell can grow, we determined the concentration to be used in the experiment for our in depth bioaccumulation characterization. Made with with BBa_K2123302 transformed in DH5-alpha and inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and overnight growth. Then, an aliquot of 100μl was taken and inoculated in three erlenmeyers with 50 ml of LM.The samples were incubated under 37°C at 150 rpm on shaker, and the optical density was measured every hour until it presented 0.6abs (measured on spectrophotometer at 600 nm wavelength). At that point 5 ppm of HgCl2 solution was added to the sample. After 8 hours of growth, the three of them were centrifuged at 12000g for 3 minutes and the supernatant recovered (LM medium).

To measure bioaccumulated Hg, we needed to quantify the Hg in the medium after incubation/exposure time. So we collected and measured the amount of Hg in LM medium supernatant recovered. In order to do so, we used the equipment Direct Mercury Analyzer (DMA-80). We used a control estimated by the DMA-80 machine.

The graph represented on Figure 3 shows the amount of Hg in supernatant (LM medium recovered) of our DH5-alpha transforming with BBa_K2123302. In the 5 ppm Hg concentration our Phytochelatin, accumulated 69% of total Hg amount in just 08:00 hours of incubation!!!

Notebook

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