Revision as of 05:44, 23 October 2016

HyaA -> E. coli

HyaA is the small subunit of hydrogenase 1 (Hyd-1, from MBH family) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structural subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaA is the first gene of hya operon, sharing the last few base pairs with hyaB. Under carbon starvation upon entering stationary phase, expression of the hya operon, which encodes Hyd-1, is regulated by sigma-38. The small subunit exhibits sequence homologies to subunits of NADH: ubiquinone oxidoreductase (Complex I). This small subunit of this [Ni-Fe] hydrogenase contains 4 Fe-S clusters ([Fe3S4], [Fe4S4]d, [Fe4S3]) that play a role in ET to and from the large subunit for H2 reduction/oxidation. The supernumerary cysteine in the proximal cluster of the small subunit also plays a role in O2 tolerance.

Usage and Biology

This part HyaA encodes for the first gene of Hydrogenase 1 gene cluster in E.coli.

Figure 1. The Hya operon on the genome of E.coli.

Characterisation of HyaA

First the recombinant plasmid with genes encoding E.coli Hydrogenase 1 was constructed. This enzyme is encoded by hya operon consisting of six genes named hyaABCDEF (BBa_K1958001, BBa_K1958002, BBa_K1958003, BBa_K1958004, BBa_K1958000, BBa_K1958006) on the genome of E.coli. The sequence was PCR amplified according to the sequence from our vessel E.coli strain BL21(DE3). We have completed the plasmid pET28a with hya cluster promoted by a T7 promoter. The following figure shows the result of enzyme digestion assay of recombinant plasmid in which our target gene cluster displayed a band around 5.5kb, which indicated successful hydrogenase expression.

Figure 2. Enzyme digestion assay of recombinant plasmid, showing the band hyaA-F. (B) SDS-PAGE analysis of recombinant E.coli, showing the band of purified HyaA and overexpressed hydrogenase in recombinant strain.

To detect induced expression of Ec-Hyd1 we performed an SDS-PAGE assay. Note that only the small subunit HyaA had a His-tag for purification on this plasmid. The purified subunit ran as the reference for hydrogenase. The assay showed that recombinant strain overexpressed hydrogenase compared to E.coli with empty pET28a plasmids.

Figure 3. Qualitative test of hydrogenase under anaerobic conditions.

We determined that our enzyme is effective using both qualitative and quantitative tests under anaerobic condition. Solutions for reaction were flushed with nitrogen and then reaction cells were vacuumed and sealed tight. The qualitative test was done after 20h of anaerobic culture (Figure 3A). We found that the recombinant strain overexpresses hydrogenase produces more bubbles than control group. We assumed that this was because more hydrogen evolves in experiment group. WO3 is a redox dye which determines the existence of reduction force in the environment with a color change to blue, compared to its original green. Again in 20h of anaerobic culture (Figure 3B), the WO3 powders displayed a darker color in the recombinant strain than that of native E.coli strain. We assumed that recombinant strain has created more reduction force than native E.coli strain.

Figure 4. Gas chromatography quantitative test of hydrogenase under anaerobic conditions.

To measure the exact quantity of hydrogen produced in 20h of anaerobic culture, a gas chromatography (GC) test on samples taken from reaction flask headspace was done accordingly later. Nitrogen was the gas carrier and bulk H2 ran as the reference, which showed a peak at approximately 1.4 minute. According to our result, the control group produced 0.58% hydrogen in headspace under 1.96% oxygen proportion which marked the level of native fermentation of E.coli. The recombinant group obviously produced more hydrogen which made up 1.25% in the headspace under 2.06% oxygen level. This doubles the production of native fermentation, indicating that our enzyme is effective under anaerobic conditions.

Sequence and Features