Proteorhodopsin
Expression and Purification
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 antibiotic. Cells were grown up to an OD600 nm of 0.6. The pellet was resuspended in fresh LB and the culture induced overnight at 37 °C with 5 mM arabinose and 10 uM all-trans retinal.
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
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 antibiotic. Cells were grown up to an OD600 nm of 0.6. The pellet was resuspended in M9 Minimal Media and the culture induced for 5 hours at 37 °C with 5 mM arabinose and 10 uM all-trans retinal.
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 by Agilent 8453 UV-visible spectrophotometer using quartz cuvette.
Proton-pumping measurements
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 supplemented with antibiotic. Cells were grown up to an OD600 nm of 0.6.
The pellet was resuspended in M9 Minimal Media and the culture induced for 5 hours at 37 °C with 5 mM arabinose and 10 uM 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 apposite 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 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 (100 ul) was taken from the sample and 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 ug/ml). After culture absorption, the plates were incubated overnight at 37 °C. The day after the number of colony plated was 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 uM all-trans retinal. 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 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. After16 h a 1:100 dilution was made in LB plus 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 uM 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 centrifuge 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 1 mL 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 adapted. The extracted pigments were concentrated with N2 and the volume was adjusted with MeOH up to 500 ul. 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 10μM of Nicotinic Acid (NA). After 20 h of induction, 0,5 mL culture containing 10^8 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. Formula used:
Proteorhodopsin Microbial Fuel Cell (pMFC)
Berkley bacteria growth
Engineered bacteria ( E. coli BL21) with ccm and MtrCAB from Berkley were grown in LB with Kanamycin (50 μg/mL) and Chloramphenicol (34 μg/mL) until OD 0.6. Cell pellets were resuspended in M9 Minimal Media until OD 1.0.
pMFC assembly
Standard protocol to assemble a Trento 2015-UNITN iGEM team Microbial Fuel Cell. Components:
- Plexiglas pieces for the chambers (4 p.)
- sheath (4 p.)
- Carbon cloth electrode (2 p.)
- tinned copper wire (2 p.)
- Proton Exchange Membrane (1 p.)
- screw (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 put the Proton Exchange Membrane with the other two sheaths like a sandwich. At the end close the MFC with the screw and fix the bolts.
pMFC data logging
Protocol standard to evaluate electrochemical parameters of a Microbial Fuel Cell. Bacterial cultures are grown and induced as described in the protocols.
- Fill the anode with bacteria and the cathode with water
- Connect the MFC to an outer variable resistance
- Connect the outer variable resistance to a voltmeter in parallel
- Open the voltage data logging software and set the parameters that the experiment needs (take a measurement each minute for 16 hours is the standard).
- Start data logging and record the data overtime.
- Once finished the test disassemble the MFC and wash it.