Difference between revisions of "Team:Nanjing-China/Proof"

Our project is to construct an artificial solar-driven system in model organisms. There are two main problems to be solved when we artificially construct solar-driven systems: media for electron transportation and enzymes to harvest electrons to chemical bonds. We successfully solved the two problems in E.coli and tested the efficiency of our system using hydrogen production.

To be specific, we used hydrogenase to harvest electrons to hydrogen bonds. We assembled six basic parts (BBa_K1958001, BBa_K1958002, BBa_K1958003, BBa_K1958004, BBa_K1958000, BBa_K1958006), which encode for EcHyd-1, into a Biobrick device and successfully expressed it in E.coli BL21 and tested its hydrogen production function. Besides, as hydrogenase 1 is oxygen intolerant, we tested its hydrogen production efficiency under anaerobic conditions at first, and came up with a unique silicon encapsulation method to maintain its hydrogen production efficiency under aerobic conditions.

To solve the problem of electron transduction, we fused PbrR (BBa_K1958007), which is another basic part of our project this year, with a previously existing basic part OmpA (BBa_J36836). PbrR is a kind of metal binding protein which can adsorb cadmium ions. OmpA is one of the outer membrane protein of E.coli. By fusing these two proteins together, we can construct a Biobrick device that can display PbrR onto the surface of E.coli so that cadmium ions can be fixed! When we add S2- into the culture medium, CdS can be fixed firmly onto the surface of E.coli and conduct electron transportation into the bacteria. The detailed experiment results are listed as follows.

Figure 1 hya gene cluster on the genome of E.coli

To overexpress hydrogenase in E.coli, first the recombinant plasmid with genes encoding E.coli hydrogenase 1 was constructed. This enzyme is encoded by hya operon where lie in all 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 plasmid construction.

Figure 2 (A) 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 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

To determine that our enzyme is effective, we used both qualitative and quantitative tests. Under anaerobic conditions. 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 condition

To measure the exact quantity of hydrogen produced under 20h of anaerobic culture, a gas chromatography (GC) test on samples taken from reaction flask headspace (600mL) was done accordingly later. Nitrogen was the gas carrier and bulk H2 was run as the reference, which showed a peak at approximately 1.4 minute (Figure 4A). A standard curve (Figure 4B) was completed to measure exact quantity of hydrogen in the samples. According to our result, the control group (Figure 4C) produced 6.88μmol hydrogen in headspace under 0.5% oxygen proportion. This marked the level of native fermentation of E.coli. Compared to control group the recombinant group (Figure 4D) obviously produced more hydrogen, which is 14.71μmol in the headspace under same oxygen level (Figure 4E). This is double the production of native fermentation which thus proves that our enzyme is effective under anaerobic conditions.

Figure 5 sketch map for MV photocatalytic reduction

Second the PbrR (BBa_K1958007) based artificial PS system is constructed according to our design. The first step to verify an artificial PS system is to test its photocatalytic capability. The redox dye methyl viologen (MV) is a well-establish indicator. In a photocatalytic reaction, photons spark excited electron on the semiconductor which then reduce MV from its +2 oxidized state to +1 reduced state (Figure 5). Increased concentration of reduce MV leads to an increase of absorbance at 605nm.

Figure 6 photocatalytic reduction of MV by TiO₂ and induced CdS nanoparticles

MV reduction was confirmed with TiO₂ nanoparticles as the positive control. The reaction mixture consist of 100mM Tris-HCl (pH 7), 150mM NaCl, 5% glycerol, 100 mM ascorbic acid and 5mM MV₂₊ with or without TiO2 particles. Light irradiation resulted in an absorbance peak around 2 minute after initiation (Figure 6A). The reaction was fast and completed within ten minutes as absorbance drops to horizontal because of the exhaustion of sacrificial electron donor ascorbic acid and the oxidation of MV+ by air oxygen.

To confirm the capability of our CdS system based on PbrR we conducted the same photocatalytic assay. Bacteria were divided into three groups. Bacteria were induced to express OmpA-PbrR protein and cultured with both Cd2+ and S2- in the experiment group. Groups that either lacked induced expression or necessary ions to build semiconductors were the supposed negative control. We found that illumination resulted in a same increasing trend in experiment group (Figure 6B). This confirmed the photocatalytic capability of our PbrR based precipitation of semiconductors.

Figure 7 gas chromatography test on hydrogen evolution of hydrogenase with CdS particles

To determine the CdS system is as well compatible with hydrogenase, we measured H2 evolution amount with CdS accompanied hydrogenase. The formation of in situ CdS nanoparticles lead to improved H2 production marking 1.22μmol in the head space (123mL) against 0.98μmol in the control group (Figure 7C).

Figure 8 TEM image of native E.coli cells (right) and cells with in situ formed CdS nanoparticles (left)

To confirm exact nanoparticles on the cell membrane of E.coli, we revealed particle clusters on the surface of E.coli using transmission electron microscopy. Black dots revealed under TEM indicate particles of size smaller than 10nm (Figure 8 left), compared to the smooth outer membrane of native E.coli cells (Figure 8 right).

Third we confirmed the stability and viability of our encapsulation method and obtained microscopic images of bacteria microballs.

Figure 9 growth curve of native E.coli and E.coli@SiO₂

To test the stability of encapsulation we drew the growth curve of both native and encapsulated bacteria. When bacteria is restricted within silicon coat, they stop multiplying which is then detected by measuring absorbance at 600nm (OD600). We found that under a 10-layer shell in the encapsulated group, the absorbance remained same for more than 12 hours in the growth curve (Figure 9). This proved that our encapsulation coat was stable for at least 12 hours which we assumed abundant for hydrogen production.

Figure 10 fluorescence decay of encapsulated E.coli by (A) absolute and (B) percentage

As the induction using IPTG is performed AFTER the encapsulation operations, living bacteria within the shells are required. The viability of encapsulation was tested through fluorescence decay and we found that silicon coats actually preserved the bacteria. Bacteria in the experiment were cultured in de-ionized water (without any nutrient) and induced to express red fluorescence (mRGF) as an indicator of the viability of bacteria cells. This experiment last for 20 days and fluorescence decayed fast in native E.coli while color remained in the experiment group (Figure 10). The closed system did not choke cells to death; the encapsulated bacteria is living bacteria.

Figure 11 fluorescent microscopic image of (A) native and (B) encapsulated E.coli cells

To further determine the structure of bacterial clusters, we obtained images of E.coli cells under fluorescent microscope (Figure 11). Cells were induced to produce mRGF fluorescent protein as signals. Bacteria from native E.coli group were scattered across the field of view however encapsulated cells formed microballs though with a slightly irregular shape. We speculated that this was because E.coli cells are not perfectly round.

At last as a proof we combined the three module: hydrogenase, semiconductor and encapsulation together to build a whole cell-based hydrogen production factory. Bacteria cells were cultured, induced to construct CdS particles, wrapped into silicon coat, induced to synthesize hydrogenase and incubated under illumination in air for 40 hours. We also conducted the same experiment but using TiO2 as positive control.

Figure 12 gas chromatography result of encapsulated E.coli

After 40h of incubation, the amount of hydrogen from the headspace in our encapsulated group with CdS measured 0.25μmol according to gas chromatography result (Figure 12). Either unencapsulated E.coli or encapsulated E.coli without CdS produced less hydrogen. The experiment was backed with 16 groups with four variables (encapsulated or not, aerobic or not, CdS precipitaion induced or not, using LB or buffer in photocatalytic reaction) to assure effectiveness of every module part: hydrogenase, semiconductor and encapsulation. This proved our success in “in air production” of hydrogen.

We succeeded in “in air production” of hydrogen. Till now we have successfully tested our hydrogenase, constructed and tested our solar driven system, designed, examined our encapsulation protocol and tested our combined design. This is a zero-to-one progress to solve the problems of both artificial PS system and oxygen intolerance against oxygen with our new system!

Now that our two systems (the artificial photosynthetic system and encapsulation system) proved to be successful, we can also expand the applications of our artificial photosynthetic system into other model organisms such as B. subtilis and yeast simply by replacing OmpA with TasA/CotC in B. subtilis and GCW21 in yeast, as all these proteins are cell surface display proteins (Figure 8). Besides, we can also expand our encapsulation system into other oxygen-intolerant enzymes to make them work under aerobic conditions.

Figure 13 Expansion of our artificial photosynthetic system into other organisms.

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