Team:Macquarie Australia/Design
DESIGN
Identifying and Researching the Problem
Nations across the world are increasingly turning towards renewable and environmentally friendly energy to power our homes, our cars and our future. Hydrogen gas is one of the renewable alternatives to fossil fuel-based energy, and benefits from having a high energy density (33 kWh/kg). When hydrogen gas is converted to electricity using a PEM fuel cell the only by product is water. Because of hydrogen's clean conversion to electricity and high energy density it is being explored as a renewable energy source by countries including Japan and Australia[1].
The world is in urgent need of a modern hydrogen gas sensor: one that is not only readily available, but also environmentally sustainable. To ensure safe use of hydrogen there is a need to have simple and reliable methods of hydrogen detection. These sensors could be deployed in a wide variety of industries which handle hydrogen gas, as standard gas detectors in homes, or to be used as a safety device for public servants such as firefighters.
Interviews with several organisations that deal with hydrogen gas including the Fire and Rescue NSW department have given us valuable insights into the problems faced in the use of current hydrogen gas detectors. Current hydrogen gas sensors are prone to false-positive results due to cross-sensitivity with other volatile substances [2]. The future hydrogen industry would benefit from a cost effective and specific hydrogen gas.
To learn more about existing hydrogen gas detecting technology, we conducted extensive literature based research about readily available sensors and technologies in development. Interviews with various stakeholders and industries who use hydrogen gas were also organised. We recorded and reviewed all feedback and information regarding the hydrogen gas sensors and the market for them.
Brainstorm for System
Our team began brainstorming ideas to create a hydrogen biosensor. We discovered several types of sensory hydrogenases, in particular, the NiFe 2c hydrogenases which have a cyclic-di-GMP phosphodiesterase/diguanylate cyclase subunit. A possibility was utilising the cyclic-di-GMP metabolite in combination with a cyclic-di-GMP riboswitch. Using these two components, we engineered a system that would detect hydrogen and produce the reporter fluorophore, eGFP. The first component is a hydrogenase which may enable production of cyclic-di-GMP (diguanylate cyclase) or degradation of cyclic-di-GMP (cyclic-di-GMP phosphodiesterase), depending if hydrogen is bound to the active metal site of the large subunit. The second component is a cyclic-di-GMP riboswitch coupled with a fluorophore, together responding to changes in cyclic-di-GMP and producing a fluorescent signal. It is assumed that higher fluorescence is indicative of higher hydrogen concentrations present. We designed a preliminary prototype containing our biosensor based on a HTS assay as shown in (Fig. 1). Using the 96 well plate format, we anticipate our biosensor can be integrated into existing systems such as a gas pipeline (Fig. 2).
Preliminary Design
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| Figure 1:HTS assay for the detection of hydrogen gas produced by microorganisms [3]. The hydrogen biosensor can easily replace the catalyst and dye, and its location above the samples could enable detection of hydrogen gas as it is released from aqueous solution. | Figure 2: Large scale preliminary prototype device to implement our system in the hydrogen production industry. |
Development and Prototype Design (Our Solution):
We are creating a hydrogen biosensor composed of two components: the sensor and the response. The sensory component contains the hydrogenase-phosphodiesterase part and the response component contains the riboswitch leading to the expression of eGFP. The sensor and response work by communicating through a secondary messenger, cyclic-di-GMP.
The Middle Man (Secondary Messenger)
Cyclic-di-GMP: A secondary messenger in most bacterial cells [4]. Primarily regulated by stress response genes (RpoS σS) and growth phase [5]. Cyclic-di-GMP upregulates biosynthesis of cellulose and curli fimbriae (a main biofilm components) [5].
Sensory Component
Hydrogenase/Phosphodiesterase (with constitutive promoter: BBa_ J23100): The hydrogenase part is a three subunit protein complex (a large subunit, a small subunit and a diguanylate cyclase/cyclic-di-GMP phosphodiesterase). The third subunit can behave as either a diguanylate cyclase (producing cyclic-di-GMP) or a cyclic-di-GMP phosphodiesterase (degrading cyclic-di-GMP)[5][6]. The activity of this domain is modulated by the binding of molecular hydrogen to the hydrogenase subunit.
The hydrogenase that we are using in our design is the Nickel-Iron [NiFe] class 2C hydrogenase from Magnetospirillum magneticum [6][7]. We picked this hydrogenase as research has identified one of the genes in the hydrogenase operon as being a maturation protease which cleaves the C-terminus after the Nickel insertion [6]. These proteases are specific for every hydrogenase.
The hydrogenase-phosphodiesterase is then coupled with a constitutive promoter.
Figure 3: Represents the NiFe 2C hydrogenase from Magnetospirillum magneticum comprised of the 5 kb operon containing small subunit, large subunit, maturation protease and cyclic-di-GMP phosphodiesterase.
Response Component
Riboswitch: Our system utilises the cyclic-di-GMP riboswitch from Candidatus Desulforudis audaxviator, which was selected as it has a known structure and sequence. Riboswitches are mRNA tertiary structures which can regulate gene translation through binding to an effector molecule [4][5]. By binding to the terminator stem of the riboswitch, ligands can prevent the rRNA complex from forming and translating the mRNA sequence [5]. In our case, the effector molecule is cyclic-di-GMP, a secondary messenger in most bacterial cells, primarily involved in biofilm formation cascade [4][5].
In our design, downstream of the riboswitch coding sequence is the eGFP reporter protein and the ribosome binding site. Therefore, the translation of eGFP will be negatively correlated to the intracellular concentration of cyclic-di-GMP. The riboswitch responds to cyclic-di-GMP concentrations to either produce or not produce eGFP. Cyclic-di-GMP binds to the aptamer domain of the riboswitch and forms a terminator stem. A terminator stem of the riboswitch could prevent translation of downstream sequences [4].
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| Figure 4: Riboswitch and cyclic-di-GMP (secondary messenger) interaction. [Bound] | Figure 5: Riboswitch and cyclic-di-GMP (secondary messenger) interaction. [Unbound] |
Testing the System
Phosphodiesterase: We tested the function of phosphodiesterase part with a Biofilm Assay. The intracellular cyclic di-GMP is positively correlated with biofilm formation in a bacterial cell, therefore by measuring the biofilm adherence capacity, we attempted to measure the cyclic-di-GMP concentration, which is inversely correlated with pde activity. The assay allows us to understand the catalytic activity of the hyd-pde complex.
The biofilm assay went through several iterations in order to view a distinct difference in biofilm production between cells with and without an active phosphodiesterase. We tested growth with different temperatures, incubation periods, with and without antibiotics, growth in minimal and high nutrient media, and different concentrations of IPTG.
We also transformed this part in different strains of E. coli - DH5a vs. Nissle 1917 - in order to see difference in biofilm production. Cells were spotted onto LB agar plates supplemented with Congo Red and Coomassie Blue, and visualised with Blue Light LEDs.
Hydrogenase: It was our goal to test the interaction between the hydrogenase and hydrogen gas. To test the functionality of the hydrogenase part HydA of M. magneticum [NiFe] hydrogenase, we measured the hydrogen saturation and desaturation rates (mV/s) in water, HydA7 (DH5ɑ +hydrogenase) and DH5ɑ (-hydrogenase) using a Clark-type electrode sensor. HydA7 was used in the experiment as it had a high level of expression. The saturation rate in water was the highest, followed by HydA7 and DH5ɑ. Similarly, the desaturation rate in water was the highest, followed by HydA7 and DH5ɑ. There was no hydrogen production when glucose was added, which confirmed the hydrogenase operates in the direction of oxidising hydrogen .
Riboswitch: We constructed two plasmids containing the coding sequences both the riboswitch/eGFP operon (either R ON and R OFF) under the stationary phase promoter and the yhjH gene under the Lac promoter, referred to as yhjH + R ON [BBa_K3151029] and yhjH + R OFF [BBa_K3151030] respectively. We transformed these plasmids into DH5α cells to perform fluorescence assays using the BMG Pherastar plate reader to measure eGFP (Ex485 nm Em520 nm) and OD (600 nm).
References
[1] Ozawa A, Kudoh Y, Murata A, Honda T, Saita I, Takagi H. Hydrogen in low-carbon energy systems in Japan by 2050: The uncertainties of technology development and implementation. International Journal of Hydrogen Energy. 2018 Sep 27;43(39):18083-18094.[2] Raj VB, Nimal AT, Parmar Y, Sharma MU, Sreenivas K, Gupta V. Cross-sensitivity and selectivity studies on ZnO surface acoustic wave ammonia sensor. Sensors and Actuators B: Chemical. 2010 Jun 3;147(2):517-24.
[3] Schrader P, Burrows E, Ely R. High-Throughput Screening Assay for Biological Hydrogen Production. Analytical Chemistry. 2008;80(11):4014-4019.
[4] Sudarsan N, Lee E, Weinberg Z, Moy R, Kim J, Link K et al. Riboswitches in Eubacteria Sense the Second Messenger Cyclic Di-GMP. Science. 2008;321(5887):411-413.
[5] Hengge R. Principles of c-di-GMP signalling in bacteria. Nature Reviews Microbiology. 2009;7(4):263-273.
[6] Greening C, Biswas A, Carere C, Jackson C, Taylor M, Stott M et al. Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival. The ISME Journal. 2015;10(3):761-777.
[7] Søndergaard D, Pedersen C, Greening C. HydDB: A web tool for hydrogenase classification and analysis. Scientific Reports. 2016;6(1).
