Team:UNITN-Trento/Experiments

Proteorhodopsin

Expression and Purification

Steps to isolate the protein:

6,000 x g centrifugation for 10 min at 4°C
pellet resuspension in Buffer A (50 mM Tris-Cl pH 8.0 and 5 mM MgCl₂)
sonication
10,000 rpm centrifugation for 20 min at 4 °C
supernatant 100,000 x g ultracentrifugation for 3 hours at 4 °C
pellet resuspension in Resuspension Buffer (100mM Phosphate buffer pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 5% glycerol, 0.3% Triton).

Anaerobic growth

After 5 hours of induction cells were split in the anaerobic workstation in dark and light condition and placed in sealed glass bottles with a rubber septum. The OD₆₀₀ nm was constantly monitored for 82 hours from when the anaerobiosis was set. Light was provided by a white halogen lamp (160 W) outside the thermoshaker set at 37 °C at 190 rpm. The optical density was measured with Agilent 8453 UV-visible spectrophotometer.

Proton-pumping measurements

The pellet was resuspended in M9 Minimal Media and the culture induced for 5 hours at 37 °C with 5 mM arabinose and 10 μM all-trans retinal. Samples were then placed in the solar mimicking apparatus to grow in light or dark conditions. pH was measured every 6 h, in a 24 h range.

Solar mimicking apparatus

An apparatus was built to test and characterize proteorhodopsin, simulating light or dark conditions. Grown cultures, during or after induction, were maintained at ~37 °C with magnetic stirring using a laboratory stir plate. The light was provided directly on cells cultures by a 160 W halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foils.

Toxicity test by serial dilutions

An aliquot of cells (100 ul) was used to do serial dilution in fresh LB, the process was done from a 102 to a 107 dilutions. 10-150μl of each sample dilution was plated on LB agar + Chloramphenicol (34 μg/ml). The plates were incubated overnight at 37 °C. The day after the number of colonies plated were counted.

To estimate the number of cells the CFU/ml was calculated as follows: $CFU/ml = \frac{\,colonies\,counted\,\cdot\,dilution\,factor}{culture\,plated\,[ml]}$

ATP assay

A single colony of transformed E. coli NEB10β was grown in 5 ml of LB broth with the proper antibiotic and incubated at 37 °C overnight. After 12-16 hours a 1:100 dilution was made in LB broth supplemented with Chloramphenicol. Cells were grown up to an OD₆₀₀ nm of 0.6. The pellet was resuspended in fresh LB and the culture induced for 5 hours at 37 °C with 5 mM arabinose and 10 μM all-trans retinal. After 5 hours of induction cells were split in the anaerobic workstation in dark and light conditions and placed in sealed glass bottles with a rubber septum. The cultures were grown overnight under anaerobic conditions. The day after a commercial ATP assay from Sigma was performed (ADP/ATP Ratio Assay Kit, MAK135), following the instructions described in the technical bulletin. The assay consists of three luminescence measurements to calculate ADP/ATP ratio.

15-15' Beta-carotene dioxygenase (BLH)

Cell growth

One colony of transformed E. coli NEB10β was grown in 5 ml of LB broth with 5 µl of appropriate antibiotic (1:1000) and incubated at 37 °C. After 16 h a 1:100 dilution was made in LB with the antibiotic. Cells were grown at 37°C until an OD₆₀₀ nm of 0,6 was reached. The culture was induced with 5 mM arabinose and supplemented with 5 μM FeSO4 and 10 mM ascorbic acid.

Extraction of pigments

100 ml of cells were grown as described above. The culture was induced with 5 mM arabinose for 24 h at 37°C. After incubation cell pellets were collected by centrifuging at 6000 rpm for 10 min at 4 °C. Supernatant was discarded and 2,5 ml of acetone were added. Sample was vortexed and incubated at 50 °C for 10 min. Another centrifuge was performed to separate the extract from the pellet, the extract was then transferred into a new vial.

TLC

Silica gel was used as the direct phase. A small sample quantity using a capillary was applied on the TLC plate. A solution of n-exane and 1% of ethyl-acetate was used as mobility phase.

UV-VIS

Extracted pigments in acetone were placed in a 700 μL quartz cuvette and the absorbance was read with an Agilent Cary 8454 UV-Vis between 300 nm and 800 nm. The samples were blanked with acetone. The reference was done with pure retinal and pure βcarotene.

HPLC

For chromatographic separation and carotenoids analysis by HPLC a reverse-phase method was used. The extracted pigments were concentrated with N2 and the volume was adjusted with MeOH up to 500 μl. The samples were injected with a syringe on a C 8 column. The reference was done with pure retinal and pure βcarotene. See following table for column and method specifications:

Column	3,5 µm Eclipse XBD-C8 column (150 x 4.6 mm)
Solvent A	methanol/water (7:3 v/v) + 12 mM of ammonium acetate
Solvent B	methanol + 12 mM of ammonium acetate
Separation	Solvent gradient: time 0 min: 35% solvent B in solvent A. time 40 min: 100% solvent A
Flow rate	0.8 ml/min

Nicotinic Phosphoribosyl Transferase (pncB)

NAD/NADH quantification

A single colony of transformed E. coli NEB10β was grown overnight in 5 mL of media culture (LB or Terrific Broth) with relative antibiotic (34 μg/mL Chloramphenicol). The culture was then diluted 1:100 in fresh medium. When an OD of 0.6 was reached, cells were induced with 5mM arabinose. Some samples were tested with 10 μM of Nicotinic Acid (NA). After 20 h of induction, 0,5 mL culture containing 10⁸ cells were centrifuged and the supernatant discarded. The cell pellet was washed with cold PBS. NAD⁺ and NADH levels were calculated with a colorimetric assay using the Sigma NAD/NADH quantification kit (MAK037) following the instructions described in the technical bulletin. Quantification was based on a standard curve made with 0, 20, 40, 60, 80 pmole/well of NADH standard. The kit provides the measures of NAD⁺ levels indirectly from total levels of NAD⁺ NADH and NADH only. The absorbance was measured with a Tecan Infinite Pro 200 at 450 nm.

The formula used was:

$\frac{NAD}{NADH} = \frac{NAD_{TOTAL} - NAD}{NADH}$

Proteorhodopsin Microbial Fuel Cell (pMFC)

pMFC assembly

Standard protocol to assemble a Trento UNITN 2015 iGEM team Microbial Fuel Cell. Components:

Plexiglas pieces for the chambers (4 p.)
Sheaths (4 p.)
Carbon cloth electrodes (2 p.)
Tinned copper wires (2 p.)
Proton Exchange Membrane (1 p.)
Screws (4 p.)
Bolts (4 p.)

Before assembling the MFC wash the plexiglas component with water and soap and expose it to UV treatment (1 hour is the standard). Wash with water the carbon cloth electrode and autoclave it. Then assemble the Plexiglas pieces between two sheath. Put inside each chamber a carbon cloth electrode with the tinned copper end exposed. To divide the anode and cathode chamber of the MFC place the Proton Exchange Membrane with the other two sheaths like a sandwich. At the end close the MFC with the screws and fix the bolts.