Introduction to the Results
Proteorhodopsin
Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to all cis-retinal. The different absorption properties are due to a single amino acid, at position 105 in the retinal binding pocket. The presence of a highly conserved Gln at position 105 in BBa_K1604010 indicates that it belongs to the blue absorbing family. [3]
Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.
We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.
Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!
After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils) the cultures were split in the anaerobic chamber in light and dark conditions. The cultures were placed in the thermoshaker that was illuminated from the outside. Half of the cultures were kept in the dark and the other half were exposed to the light.
The OD600 was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.
The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (BBa_K731201). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.
While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.
Solar mimicking apparatus NEB10β cells transformed with BBa_K1604010 were grown exposed to light (left side) or in dark condition (right side). The cultures were maintained at ~37°C with magnetic stirring using a laboratory plate. The light was provided by a 160 Watt halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foil (not shown).
Since we observed that there was a possible activation of the proton pump without light, we decided that our next test would be a proton pumping experiment as described in the literature [2][5].
The ΔpH between the light exposed proteorhodopsin and the two negative controls (proteorhodopsin in the dark and araC-pBAD in the light is 0.22. This result evidenced that although there is a basal acidification of the medium due to the bacteria metabolism, our device acidifies the medium thank to the activation of the proton pump when the bacteria were light exposed.
cidification of culture medium by BBa_K1604010 A NEB10β cells transformed with BBa_K1604010 were grown until an OD600 of 0.7 was reached and induced in M9 Minimal Medium with 5mM of arabinose and supplemented with 10 uM of all-trans-retinal. The induction was done in the dark. The samples were then placed in the “Solar” apparatus with or without light. pH was measured every 6h, in a 24h range.
We improved the proteorhodopsin part that we extracted from the registry, placed under an inducible promoter and we fully characterized it to demonstrate that the proton pump doeswork when the bacteria are light exposed. This membrane protein does require retinal to properly fold and increases the lifespan and the vitality of the engineered bacteria in anaerobic conditions. We did experience some difficulties in finding the right conditions of growth, light exposure and to reach anareobiosis. Also from our experience, this is a delicate system that showed sometime variability in the measurements between different biological samples. However we optimized the system and we now have a functioning device that can be used in our MFC.
Proteorhodopsin are happy to stay in our Microbial fuel Cell under the sun. Check out our results. [link alla pag della MFC]
PncB: nicotinic acid phosphorbosyl-transferase
Our device is controlled by an inducible arabinose promoter built by the Unitn iGEM team in 2012. PncB was extracted by E. coli genome, the illegal site PstI was removed, and it was placed in pSB1C3 (BBa_K1604030). Subsequently it was placed under the araC-pBAD promoter (BBa_K1604030).
PncB enhances NAD production by ~2.5 fold
Our goal was to demonstrate that pncB increased intracellular levels of NAD and thus NADH. We quantified the levels of NAD by a colorimetric test that measures the levels of NAD indirectly by quantifying the concentration of NAD total (NAD + NADH) and NADH only. To make precise quantitation a standard curve with NADH was built. The test provides the ratio of NAD/NADH
NADtotal = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.
NADH = Amount of NADH in unknown sample (pmole) from standard curve.
BBa_K1604031 does increase NAD levels by 126% (2.5 fold) and NADH levels by 44% (1.4 fold) when expressed in NEB10β. Although we did see an enhancement in NAD levels, this did not correlate to a proportional boost in NADH levels. We plan in the future to add a NAD reducing enzyme and to give a medium able to enhance the cell metabolism to further increase NADH intracellular levels.