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Wet LabWet Lab

L.Cogli R.Bramato G.Alfarano E.Rizzo G.Lazzari D.MagrìL.Cogli R.Bramato G.Alfarano E.Rizzo G.Lazzari D.Magrì L.Cogli R.Bramato G.Alfarano E.Rizzo G.Lazzari D.MagrìL.Cogli R.Bramato G.Alfarano E.Rizzo G.Lazzari D.Magrì

M.Corvaglia L.Scoletta M.Montinaro M.Corvaglia L.Scoletta M.Montinaro M.Corvaglia L.Scoletta M.Montinaro M.Corvaglia L.Scoletta M.Montinaro

Sensing relies on nickel regulator NikR from H.pylori (HpNikR): though homologue of E.coli NikR, it has a stronger dependence on Nickel ions and a wider range of regulated elements, being a rare case of pleiotropic regulator in bacteria. As HpNikR could contemporaneously activate and repress gene expression [3-4-5], so we thought to develop a synthetic gene cluster under HpNikR-regulated promoters.

The two different bacterial populations (SENSE and STORE), communicate with each other through Quorum Sensing: we used the LuxI/LuxR system from Alivibrio fischerii. The first population senses nickel concentration in medium and translates it in a fluorescence signal shift (from GREEN to RED), then activating Acyl-Homoserine-Lactones (AHLs) production. The second population receives the QS signal and sums it up to the nickel detection signal (as in an AND gate), activating the storage and AHLs signal amplification.

The Nickel storage system is even taken from our pathogenic friend Helicobacter pylori: Hpn protein is a Nickel binding protein, whose purpose is to store the Ni2+ inside the cell and to supply them with other proteic interactors [5]. Hpn can be expressed in a heterologous model system (E.coli), increasing bacterial resistance up to a toxic dose of Nickel ions and allowing Ni2+ intracellular accumulation [6].

WikiWiki

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31)Denkhaus and Salnikow, Critical Reviews in Oncology/Hematology, 42 (2002) 35–562)Contreras et al., Molecular Microbiology, 49 (4) (2003), 947–9633)Muller et al., Nucleic Acids Research, 39 (17) (2011), 7564–75754)Abraham et al, Journal of Inorganic Biochemistry, 100(5-6) (2006), 1005-145)Seshadri et al., Journal of Bacteriology, 189 (11) (2007), 4120–41266)Ge et al., Biochemistry Journal, 393 (2006), 285–293

Nickel is one of the most widespread heavy metals in the ecosystem and it is essential for many organisms, from bacteria to higher forms of life. However, as all the heavy metals, even a small amount of Nickel higher than the essential dose could be toxic, leading to various noxious effects [1]: then the need to remove its excess from many substrates. Nowadays bacteria-mediated bioremediation from inorganic substances seems to be a considerably relevant frontier in microbial biotechnologies. Our project aims to develop a living system, in an easily monitorable bacterial platform, who would work as a Nickel detector and a Nickel remediation system. We thought to split the sense and store machineries in two different bacterial populations, linked via Quorum Sensing. The whole system is based on genetic parts from the pathogen Helicobacter pylori, whose evolution led to a stronger reliance on Nickel ions for host interaction and pathogenesis.

SupervisorsSupervisors

PURIFICATION PLANT IMPLEMENTATIONIntegrating our bacterial system in a sedimentation or oxidizer tank of a common wastewater treatment plant, would allow nickel removal without the need to add dedicated sections to the plant.

PROBIOTICS Integrating the device in Gram-negative species harmless to human health, such as Bacillus or Lactobacillus, we could obtain a probiotic product for nickel allergic people. In particular, just transforming Lactobacilli with Hpn you could create yoghurt or milk suitable for intestinal nickel removal. These microorganisms accumulate the metal at a cytosolic level through bioconcentration, then they would normally be expelled via the fecal-oral route.

ICP-AES assay: we performed ICP-AES assay (A) on purified HpNikR (BBa_K1151000). The intensity of emission is related to the concentration of nickel in our samples. The nickel incubated samples show a direct relationship between protein concentration and ion concentration.

Fluorimetric analysis: we set up this experiment to evaluate the shutdown of a fluorescent reporter signal due to NikR binding to negatively regulated promoters. We added a fixed quantity of IPTG and NiSO4 to BL21 transformed cells. The expression of the repressor, NikR (BBa_K1151000), under the control of a pLac promoter, has effects on the fluorescence level of GFP only in the cultures incubated with nickel. Repression of GFP expression is shown with both the constructs BBa_K1151036 (B) and BBa_K1151038 (C) with NikR responsive promoters. The proof shows the correct operation of the nickel-dependent repression mechanism operated by NikR on the two promoters of our design, pnikR and pexbB.

CryoTEM microscopy CryoTEM microscopy of purified HpNikRof purified HpNikR

We organized an open seminarseminar, about iGEM, synthetic biology and our project, with a wide audience participation of students, professors and local politicians. Our adventure as iGEMmers has so attracted local medialocal media.We are among the founders of BioBANGBioBANG, a new born association aiming to promote the interaction between academic world and biotech enterprises.The wet lab group of our Team took part to PlaydecidePlaydecide to discuss about Orphan Drugs and Staminal Cells, an event organized for the European Biotech WeekEuropean Biotech Week.

HpNikR NICKEL- AND DNA- BINDING ACTIVITY CHARACTERIZATION

Ni2+-BINDING RELATED PROTEIN STRUCTURAL CHANGES CHARACTERIZATION ATR-FTIR spectroscopy assay:the analysis of the spectrum (D) of the native protein shows two peaks at wavenumber of 1261cm-1 and 800cm-1; these peaks are absent in the protein incubated with Ni2+ and everything is confirmed by the difference spectrum. These peaks could be assigned to a tyrosine residue deprotonation due to the interaction with nickel. So it can be assumed that the bond to the metal induces a change necessary to the regulator NikR for the interaction with DNA.Raman spectroscopy assay:with Raman spectroscopy analysis it is possible to state that in the 1000-1600 cm-1 region the difference in signal comes from amide groups (I, II e III). Instead, to higher frequences, in the 2900-3200 cm-1 region the difference in signal comes from the stretching of the CH2 and CH3 bonds.Conformational changes could be seen in the amide regions, but further investigations are needed.

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To analyze how much synthetic biology, its applications and its ethical implications are known in our academic world, we decided to submit a short survey. We submitted it to professors, researchers, doctoral students and non graduate students.We concluded that, in our academic world, people of different categories know about Synthetic Biology and its applications, though a lot of people only have some unclear ideas about this discipline. Most of them have great expectations for its applications in Human health and they think that it is important that science could take advantage of theoretical knowledge for applicative uses. For the people of our University Synthetic Biology may have better applications in the Environmental field and in the Biomedical field. We hope that our iGEM experience could help researchers and students focus on this growing research field.

Acknowledgements:

Prof. A. Danielli (plasmids); Prof. M. Benedetti, Dr. D. Migoni (ICP-AES); Prof. L. Giotta (ATR-FTIR); Prof. D. Manno (CryoTEM); Prof. A. Serra (Raman); Prof. GP. Di Sansebastiano (fluorimetric assay); Dr. D. Pacoda (safety); Dr.Eng. A. Leanza (application).

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