Team:UNITN-Trento/Results

Introduction to the Results

Proteorhodopsin

Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses light energy to generate an outward proton flux. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H+-ATP synthase complex[2].

The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the SAR86 group. The original cluster is composed of 6 genes: four are involved in beta- carotene production; one is implied in beta carotene cleavage into two molecules of retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. [3]

Schematic representation of the PR gene cluster identified in clone HF10_19P19Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.

Proposed mechanism of PR associated to the ATP-synthase complex Light-activated proteorhodopsin pumps protons outwardly, increasing the proton motive force. Protons can then reenter the cells through ATP-synthase complex, powering the ATP production.

Fig 3

Proteorhodopsin was taken from the Registry (BBa_xx) part of Caltech 2012. From the experience of Caltech 2012 we saw that they were not able to express and functionally characterize the part. We took the challenge to improve this part!

We have built two different devices to produce Proteorhodopsin and added a RBS which was missing.
BBa_K1604010. Proteorhodopsin producing device under the control of aracpBAD.
BBa_K16040xx. Device for the production of Proteorhodopsin and biosynthesis of retinal.

Retinal is the key!

We have screened several parameters (media, temperature, time of induction) to discover that the optimal expression conditions were in LB at 37C overnight in the presence of 10 μM of all-trans retinal. Attempts to express the protein in the absence of retinal failed. Proteorhodopsin is a membrane protein that needs the time to fold properly into the membrane and requires retinal to bind the pocket and help the formation of the proper folding.

The expected molecular size is 28 KDa. The SDS gel shows a band corresponding to around 37 KDa, as it was seen in other studies [4]. This is probably due to post-translational modifications.

Expression of ProteorhodopsinNEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 μM of retinal at 30°C or 37°C. Negative control were cells transformed with BBa_K731201 (i.e. araC-pBAD).

We attempted also to purify the protein from the bacterial culture by sonication followed by ultracentrifugation and we were happy to see that the purified protein was also RED, while the negative control was not.

Although LB gives the maximum expression as shown in the SDS page, we were able to successfully express Proteorhodopsin also in M9. This result was not visible by SDS page, but it is demonstrated by the presence of a bright red colored pellet typical of retinal bound to Proteorhodopsin.

M9 is the perfect culture media for our MFC, to maintain the correct proton equilibration between the anodic and cathodic chambers, and maintains a more stable signal (see our MFC results). Therefore we decided to use these growth conditions for the functional characterization.

Purification of Proteorhodopsin. NEB10β cells transformed with BBa_K1604010 and BBa_K731201 were induced in LB at 37C in the presence of retinal. The cell pellets were resuspended in 50 mM Tris-Cl pH 8 with 5 mM MgCl2 and sonicated. The lysate was centrifuged at 10,000 rpm for 20 min at 4C. The supernatant was ultracentrifuged for 100,000 g for 3 hours at 4C. The three tubes in front contain proteorhodopsin purified fractions and the three tubes in the back are negative controls treated in the same conditions

Proteorhodopsin expression in M9Cells transformed with BBa_K1604010 and BBa_K731201 were grown in LB and transferred in M9 at an OD of 0.6 and induced with arabinose with the presence of 10 µM of retinal. After 6 hours of induction the cells were centrifuged and the supernatant was discarded. From left to right: araC-pBAD induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced (C) and not induced (D) both without retinal.

Light or no light that is the question.

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]

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!

Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.

Anaerobioc growth of BBa_K1600410 E. coli transformed with BBa_K1604010 (blue line) and BBa_K731201 (green line) were grown in LB at 37C untilan OD of 0.6 and induced in M9 minimal medium with 5 mM arabinose and 10 uM retinal in the dark. After 5 hours of induction the culture were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil. Light exposed samples were excited with a 160W halogen light bulb placed outside the incubator. The blue line (proteorhodopsin) is the result of the average of 6 different samples (3 in the dark and 3 in the light) while the green line (araC-pBAD) is the average of 1 sample in the dark and 1 in the light.

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.

PncB: nicotinic acid phosphorbosyl-transferase

Increasing the levels of NADH

Our goal was to boost electron production by increasing the concentration of electron carriers (i.e. NADH). To achieve this goal we decided to engineer E. coli with an enzyme that would provide more intracellular NAD, and thus NADH.

PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. This gene is naturally found in E. coli and encodes for the enzyme NAPRTase (nicotinic acid phosphorbosyl- transferase) that catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid).

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 is not toxic if overexpressed in E.coli

NEB10β transformed with BBa_K1604030 (araC-pBAD-pncB) or BBa_K731201 (i.e. araC-pBAD) were grown up to an OD of 0.6, splitted in two tubes of 23 mL each and induced with 5 mM of arabinose. Negative controls were not induced.
The OD (600 nm) was measured every 45 minutes for 5 hours. All measurements were done for 3 different biological samples and 3 technical measures.

Although the growth rate is slightly decreased, due to the cell stress when expressing pncB, the data indicate that this enzyme does not have toxicity effect on the cells.

Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (i.e. aracpBad). Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. BBa_K1604031 (Orange line) and BBa_K731201 (green line) induced with 5 mM arabinose. BBa_K1604031 (yellow line) and BBa_K731201 (blue line) not induced.

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

NAD_total = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.
NADH = Amount of NADH in unknown sample (pmole) from standard curve.

__Title of the Figure__ NAD and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037) following the instructions described in the technical bulletin. Panel A: Standard curve (0, 20, 40, 60, 80, 100 pmole/well of NADH)). Panel B: NAD/NADH levels for three biological samples of BBa_K1604030 (green) and one negative control BBa_K731201 (blue). The cells were grown as described previously.

__Title of the Figure__ Lane B samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). Lane C samples 2-9 NAD total levels; Lane D samples 2-9 NAD total repeated with a 2 fold concentrated sample; Lane E NADH only; Lane F NADH only, repeated with a 2 fold concentrated sample. In lanes C-F the order of the samples is: 2 technical replicates of the negative control, and 2 technical replicates of each of the 3 biological samples of BBa_K1604031. The plate was read with a Tecan Infinite M-200 pro instrument at 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours to allow color development. The data shown are representative of the best measurement at 2 hours.

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.