Team:UniSalento Lecce/Overview

1 - Background

Nickel is a very widespread heavy metal and its levels in waters and other environmental matrixes are highly dependant on human activities. A lot of industrial applications lead to an increase of nickel amounts in the environment, and the growing nickel contamination led to an increase in Nickel related allergies (more than the 15% of the population suffers on nickel allergy) and other pathologies concerning the reproductive, cardiac and respiratory systems. Furthermore, Nickel compounds have been classified as cancerogenic by the International Agency for Research on Cancer (IARC, see http://monographs.iarc.fr/ENG/Monographs/vol100C/mono100C-10.pdf). The risks for human health made necessary, as for many heavy metals, to remove this metal from the environment. Nowadays nickel can be chemically removed from fluids using a chelating agent, known as H2DMG (dimethylglyoxime),which helps the precipitation of the nickel metal ions for its dosing and removal. We thought of a living platform carrying a nickel sensing device, coupled via Quorum Sensing to a second system (i.e. another bacterial population) which will remove the nickel from the environment. Our two-population system could be implemented in a water purification plant, allowing a regulated heavy metal removal without the need for chemicals (see the Applications page).

2 - Nickel sensing

The sensing device is based on a set of genetic parts from the pathogen Helicobacter pylori. This microorganism, which colonizes the stomach, requires a large amount of nickel to mantain its homeostasis in such a hard environment. Particularly, Ni++ ions are essential for Urease activity, the key enzyme for H.pylori survival in the acidic environment of the stomach, as well as for a respiratory hydrogenase. Urease makes up to 10% of the total cell protein synthesis, so that nickel homeostasis becomes an essential step for the survival of the pathogen.

2.1 - HpNikR: a pleiotropic regulator

Helicobacter controls this homeostasis through a nickel regulator, HpNikR, an homologue of NikRs from different bacterial species (including E.coli). However, in H.pylori NikR acts as a pleiotropic regulator, being a rare case of master switch regulator in bacteria. Its strong reliance on nickel concentrations and its multiple responsive elements, both positively or negatively controlled, make us think of developing a synthetic regulon for nickel sensing.

We cloned HpNikR from H.pylori strain G27 from the plasmid pET-NikR, sent us by prof. Alberto Danielli (University of Bologna), the gene was cloned using the following primers (including Biobrick Prefix and Suffix, lowercase):

nikrFor: gtttcttcgaattcgcggccgcttctagATGGATACACCCAATAAAGACG

nikrRev: gtttcttcctgcagcggccgctactagtattattaCTATTCATTGTGTTCAAAG

2.2 - HpNikR-controlled promoters

HpNikR, as said above, has multiple target promoters, some of them being upregulated, some being downregulated, responding to nickel concentration in the environment.

2.2.1 - Negative promoters

As shown above, nickel-bound NikR can downregulate the expression of various target. We obtained a genomic region from H.pylori G27 containing a divergent operon with two NikR-regulated promoters. The plasmid was sent us again by prof. Alberto Danielli as pNKTB. The promoters control the expression of the exbBD operon (for siderophore uptake, called Pexb) and for nikR expression itself (PnikR). According to functional segmentation of the divergent operon (as in Delany et al., see bibliography), we tried to separate the promoters, using the following primers (including Biobrick Prefix and Suffix, lowercase):

IntergenicaFor: gtttcttcgaattcgcggccgcttctagagTGAGAAAAATCCTTTTTTG

pnikrev: gtttcttcctgcagcggccgctactagtaTGAGAAAAATCCTTTTTTG

pnikfor: gtttcttcgaattcgcggccgcttctagagAATTCAAACGCTCTTATG

pexbfor: gtttcttcgaattcgcggccgcttctagagACTGGATTTAAATGGTTG

pexbrev:gtttcttcctgcagcggccgctactagtaGCACCCTATAAGAAGGCATC

(Here the PCR Results) So we obtained the whole divergent operon and Pexb and Pnikr. These promoters were cloned upstream of BBa_E0240, becoming parts BBa_K1151009,BBa_K1151010, BBa_K1151011. Here you can see (link to experimental data) characterization data for BBa_K1151036 and BBa_K1151038: constructs made up by the promoters cloned upstream of GFP cds together with BBa_K1151006, HpNikR coding device.

2.2.2 - Positive promoter

We also tried a positively-regulated promoter, from UreABCDE operon (PureA). We got it synthetized from IDT, obtaining BBa_K1151005. We weren't able to characterize BBa_K1151005 activity.

3 - Storage system

The second part of our project aimed to develop a bacterial population able to store nickel, basing its activity on the Hpn protein from H. pylori and a Quorum Sensing signalling: this last natural process is the basis of interaction between the two bacterial communities. The storage system of our design allows to accumulate the metal by bioconcentration: once accumulation occurred, it is necessary to dispose of the bacterial biomass containing the metal. See the Applications page for a design of purification plant implementation.

3.1 - Hpn protein

Hpn is an histidine-rich protein whose role is to store Nickel ions in the cytoplasm of Helicobacter. In conditions of Nickel starvation, this protein supplies the ions to the nickel-dependent machineries. We thought we can use this protein to make E.coli accumulate nickel. Hpn has been demonstrated not to be toxic even if highly induced in this heterologous system (Ge et al., see Bibliography). Hpn coding sequence has been synthetized by IDT, and submitted as BBa_K1151001, and its derivative translational unit BBa_K1151004, generator BBa_K1151025 and coding device BBa_K1151013. We weren't able to characterize these parts activities.

4 - Nickel-dependent Quorum Sensing Network

At first, our project was designed to work on a single plasmid, for both the sensing and metal-accumulation functions. To increase the safety of our device, giving more control to the human operator, we choose to separate the two phases of the project in two different bacterial populations. The activation of the storage depends on the activation of the sensing population. So, to integrate the two systems, we exploited the mechanism of intercellular communication of Quorum Sensing. Evaluating the effectiveness of operation of various QS BioBricks present in the Registry, we choose BBa_K805016, BBa_C0261 and BBa_R0062, from Alivibrio fischerii, based and dependent on the synthesis of AHLs as signaling molecules. In our system, the start of communication is Ni2+ and IPTG dependent, both necessary for the synthesis of HpNikR, the genic master regulator which activates the translation of LuxI, the QS inducer. The LuxI protein catalyzes the synthesis of [N-]acyl-homoserine lactones (AHLs), the signalling molecule, from S-adenosylmethionine (SAM) in the sensing population. Quorum sensing molecules reach the other bacterial population by diffusion. Here we built a system designed on the principle of positive feedback. In fact, the storage population integrates the gene LuxR within the plasmid we designed: LuxR codes for the receiver protein. Placing the transcription of the LuxI gene under the control of the promoter LuxPR, we obtained an AHL signal amplification phenomenon.

Under the same promoter and within the same synthetic operon we also placed the gene of the nickel-binding protein, Hpn, thereby obtaining a triple control of the protein synthesis process in the accumulator population, that depends upon the concentrations of Ni2+, IPTG and AHLs.

5 - General cloning and working schemes

Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.

6 - Bibliography

Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in futurum.