Team:Macquarie Australia/Description
PROJECT INSPIRATION AND DESCRIPTION
This year, Team Macquarie_Australia has chosen to develop a hydrogen gas biosensor.
A 2018 report released by the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) estimates that Australia will become the world’s primary producer of renewable hydrogen gas by 2030 [1]. It is suggested that up to 10% hydrogen gas will be mixed into the reticulated natural gas pipelines in Australia in the transition from fossil-fuel based energy production to more sustainable, emissions-free alternatives. Intrigued by the increasing use of hydrogen and ongoing success of the 2017 Macquarie_Australia iGEM biohydrogen project, our team sought interviews with representatives from industries and groups that would be directly impacted by the emergence of a world-leading renewable hydrogen gas economy in Australia, and whether they had any issues that synthetic biology could potentially solve. Our interview with the emergency response and HAZMAT unit, Fire and Rescue NSW (FRNSW), revealed that the existing hydrogen gas detection technology is outdated due to the rapid advances in renewable hydrogen gas production.
Upon consultation with researchers who are continuing the development of the 2017 Macquarie_Australia iGEM biohydrogen project, we discovered that their research could also significantly benefit from improvements in hydrogen gas detection technology. High-throughput screening (HTS) assays of hydrogen-producing strains uses expensive reagents (such as water soluble tetrazolium indicators and sulfonated Wilkinson's catalysts) for laboratory scale hydrogen gas quantification. A biosensor (or biological detector) could be easily integrated into HTS assays to easily identify and select the best hydrogen-producing bacterial strains, and allow for significantly faster and less expensive optimisation of these cultures (Fig. 1).
Figure 1: HTS assay for the detection of hydrogen gas produced by microorganisms [2]. 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.
Significant safety considerations must be addressed when large quantities of hydrogen gas are stored or transported. Hydrogen gas is naturally odourless, colourless, diffuses easily through many media, and when it is utilised for energy, no polluting greenhouse gases are emitted [3]. However, hydrogen gas is also extremely flammable and explosive at 4% concentration in the air, and when it burns, the flame is completely invisible to humans [4]. Adding odourants to hydrogen gas significantly reduced the performance of fuel cells [5]. Consequently, as hydrogen fuel cells increase in popularity throughout the globe, a reliable and accurate sensor for the detection of hydrogen gas leaks is critical [6]. For example, the method most commonly used for producing hydrogen gas involves natural gas steam reforming [7]. As the vast majority of hydrogen gas sensors have some cross-sensitivity with natural gas, this causes challenges in measuring the efficiency of making hydrogen gas by this method using normal hydrogen gas detecting equipment [8].
Compelled by the urgent need to significantly improve gas detection technologies, we aim to use synthetic biology to make Escherichia coli into a hydrogen gas biosensor, like a bacterial canary in a coal mine. Biological systems have naturally evolved to respond rapidly to changes in their intracellular and extracellular environments [9], a unique feature which may be utilised in the design of a synthetic biosensor [10]. The highly specific nature of enzymes are advantageous and as a component of a biosensor, it may only respond to a targeted compound, effectively eliminating the possibility of cross-sensitivity [11].
After extensive research and informed planning, our biosensor will consist of two parts:
1. A sensory Nickel Iron class 2c hydrogenase from Magnetospirillum magneticum. This is composed of a small subunit, large subunit, protease and diguanylate cyclase/cyclic-di-GMP phosphodiesterase. This will detect H2. 2. A cyclic-di-GMP riboswitch from Desulforudis audaxviator. This is coupled with a fluorophore reporter, enhanced green fluorescent protein (eGFP). During transcription, cyclic-di-GMP binds to the cyclic-di-GMP riboswitch, creating a terminator stem loop that prevents transcription of eGFP. The riboswitch terminator only forms when cyclic-di-GMP is bound to its aptamer. In the presence of H2, the hydrogenase activates cyclic-di-GMP phosphodiesterase, which in turn breaks down the cyclic-di-GMP, removing the terminator stem loop from the riboswitch, allowing transcription and subsequent translation of eGFP. Combination of the sensory system and fluorophore reporter will enable detection of hydrogen gas. We hypothesised a decrease in hydrogen gas in the cellular environment would cause less hydrogen gas to bind to the cyclic-di-GMP phosphodiesterase, leaving it unactivated. Cyclic-di-GMP can bind to the cyclic-di-GMP riboswitch to form a terminator stem loop, preventing translation of eGFP. This mechanism could also occur in the reverse direction, where an increase in hydrogen gas in the cellular environment would correlate with an activated phosphodiesterase, activated riboswitch and a fluorescent signal. We aim to construct our hydrogen gas biosensor through BioBrick assembly of plasmids containing individual components (Fig. 2), then transform the assembled BioBricks into E. coli. To test the functionality of parts and the assembled biosensor, we plan to use a biofilm formation assay, GFP production assay and Congo Red spot assay to examine responses to cellular stresses and observe colony morphology.
Figure 2: Representation of our plasmids combined into one final system
References
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9. Fiegna F, Moreno-Letelier A, Bell T, Barraclough TG. Evolution of species interactions determines microbial community productivity in new environments. The ISME Journal. 2015;9(5):1235-1245.
10. Plummer SM, Plummer MA, Merkel PA, Hagen M, Biddle JF, Waidner LA. Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species. Enzyme and Microbial Technology. 2016;93:132-141.
11. Peracchi A. The limits of enzyme specificity and the evolution of metabolism. Trends in Biochemical Sciences. 2018;43(12):984-996.
