Team:Macquarie Australia/Demonstrate
DEMONSTRATE
Our Hydrogen Biosensor
Our biosensor consists of two components:
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).
Figure 1: Representation of our plasmids combined into one final system
Assembly of our Hydrogenase component
The sensory M. magenticum [NiFe] hydrogenase, part BBa_K3151025, was assembled using standard assembly. We ordered hydrogenase constructs pUC57Kan-HydA (hydrogenase large and small subunits, maturation protease) and pUC57Amp-HydB (cyclic-di-GMP phosphodiesterase) from GeneWiz. They were transformed into E. coli DH5α competent cells and amplified. To confirm the presence of the two parts, we performed single (EcoRI) and double (EcoRI and PstI) restriction enzyme digestions, followed by agarose gel electrophoresis, SDS-PAGE and sequence confirmation (Macrogen). See Figures 18-20 Results Page.
Testing Hydrogen Consumption with Clark-type electrodes
The Clark-type electrodes can be used to measure dissolved hydrogen gas sample by changing the polarity of the electrodes. 1 mL of each sample was placed in the Clark-type electrode chamber. The chamber was sealed with a plug to create a dead space above the sample. The plug has a small hole in it to allow for the delivery of hydrogen gas to fill the dead space between the surface of the sample and the plug. A polarization voltage was applied to the electrode (600 mV). Hydrogen gas was applied at constant flow over the sample surface. Magnetic stirrer inside the sample chamber was set to 425 rpm. The temperature was maintained at 25 °C for all samples. The hydrogen saturation and desaturation rates are represented as negative mV per second (Figure 2A and C). Once the sample is saturated with hydrogen (Figure 2B), the hydrogen gas is disconnected, and the dead space is allowed to vent with air.
Figure 2: Clark-type electrode configured to measure dissolved hydrogen in -mV. A) maximum hydrogen saturation rate, B) maximum hydrogen saturation, and C) maximum hydrogen desaturation.
Demonstrating the Expected Hydrogenase Function
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 (negative control [-hydrogenase]) using a Clark-type electrode sensor. HydA7 signifies the hydrogenase (HydA), and the 7th biological replicate sample. The results showed a significant difference between the DH5ɑ and HydA7 bacterial cells in their rates of hydrogen absorption.
The hydrogen saturation and desaturation rates (fig. 3, fig. 4), are represented as mV per second. The saturation rate in water was the highest, followed by HydA7 and then DH5ɑ (fig. 3). Similarly, the desaturation rate in water was the highest, followed by HydA7 and the lowest, DH5ɑ (Fig. 4). The three samples represented in each graph are water, DH5ɑ (-hydrogenase), and (DH5ɑ +hydrogenase).
Figure 3: Maximum hydrogen saturation rates of water (standard), DH5α (negative control), and DH5α HydA7 transformant. Error bars are +/- StDev, n=2.
Figure 4: Maximum hydrogen desaturation rates of water (standard), DH5α (negative control), and DH5α HydA7 transformant. Error bars are +/- StDev, n=2.
Water is the standard, representing the maximum hydrogen saturation rates. When comparing the DH5ɑ with and without hydrogenase, a clear difference is demonstrated in the maximum rate of H2 saturation (mV/s) (fig. 3). H2 rate of consumption is higher in our HydA7 sample compared to untransformed DH5ɑ cells. There is overexpression of hydrogenase in the DH5ɑ HydA7 sample, therefore, in the presence of hydrogen, the HydA7 cells appeared to bind hydrogen, thus allowing for more hydrogen to be absorbed into solution before its maximum saturation is reached. This may be specifically attributed to the hydrogen binding in the hydrogenase active site. Conversely, the DH5ɑ cells without the hydrogenase did not appear to absorb as much hydrogen, resulting in lower hydrogen saturation rate. This result demonstrates that there is significant hydrogen binding in hydrogenase binding site of our modified HydA7 cells
A clear difference when comparing the DH5ɑ with and without hydrogenase is demonstrated once more with rate of H2 desaturation (fig 4). When comparing the data for DH5ɑ (-hydrogenase) and our DH5ɑ (+hydrogenase), the desaturation rate for the HydA7 is higher compared to that of the DH5ɑ cells without the hydrogenase, demonstrating the ability of the hydrogenase to bind hydrogen.
Hydrogenases are known to be reversible, where they can both produce H2 or Detect H2. In order to demonstrate that our hydrogenase BBa_K3151025 does not function as a H2 producer, we performed a hydrogen electrode test comparing it to the hydrogen producing gene cluster (HPGC)(fig. 5). This gene cluster was taken from the Macquarie Australia 2017 iGEM team. In black, is represented by E. coli (+HPGC) and in blue, is represented by E. coli (+Hydrogen sensor). The clear indication of hydrogen gas production can be seen by the HPGC, as opposed to the HydA7 (Hydrogen Sensor), which shows no indication that any hydrogen gas is being produced. This result shows that our [NiFe] hydrogenase taken from M. magneticum operates only in the direction of oxidizing hydrogen, and that the oxidation is not reversible.
Figure 5: Hydrogen production in DH5α HydA7 transformant following addition on 20 mM glucose.