Cultivation of the strains
1. 30 ml of complex medium for H. volcanii are inoculated with a plasmid-containing strain in at least three biological replicates. Additionally a culture with an empty vector is grown as a negative control.
2. After reaching stationary phase, these cultures are used to inoculate a culture of 30 ml fresh medium and cultivate the cells to mid-exponential phase (about 4*108 cells/ml).
3. Then the mid-exponential cells are used to inoculate a culture of 30 ml fresh medium that is cultivated again. The resulting mid-exponential cultures are harvested and used for the assay and Northern blot analysis.
Harvesting the cultures
4. For Northern blot analysis, at least two 2 ml aliquots of mid-exponential cultures are removed and centrifuged (13.000 rpm, 2 minutes, room temperature). The supernatant is discarded, and the pellets can be stored at -80°C and used later for RNA analysis as described in the method “Northern blot analysis” (https://doi.org/10.21203/rs.2.11264/v1).
5. For the AraDH enzymatic assay, 20 ml of the cell cultures are removed and samples are centrifuged (4000 rpm, 15 minutes, 4°C). The supernatant is discarded and the resulting pellets are resuspended in 5 ml basal salt.
6. The cells are collected by centrifugation (4000 rpm, 15 minutes, 4°C) again, the supernatant is discarded and the resulting pellets are suspended in 1 ml basal salt.
7. To break open the harvested cells, sonication is used. The cell suspensions are kept on ice during the process and sonication is performed three times for each 30 seconds (duty cycle 50%, output control 3) and one-minute cooling period.
8. After lysis, the samples are centrifuged (13.000 rpm, 30 minutes, 4°C) to remove the cell debris, and the supernatant is transferred to a new tube. The resulting cytoplasmic extract is used for the enzyme essay.
Determination of the specific AraDH reporter enzyme activity
9. The AraDH enzymatic assay is performed in a 96 well plate. First, the MTP photometer is warmed up to 37°C. After that, different cytoplasmic extract dilutions (1:5, 1:10) are made and measured each in two technical replicates.
10. For each sample, a reaction mix was made, that contained 100 µl cytoplasmic extract dilution, 150 µl 3 M KCl (preheated to 40°C) and 25 µl arabinose solution. The reaction is started by adding 25 µl NADP+-solution.
11. After starting the reaction, the optical density at 340 nm is measured with the photometer for 20 minutes, with measure points each 20 seconds.
12. To analyze the volume activity of AraDH, dilutions are chosen, which have a linear increase of extinction for at least 180 seconds (ΔE).
13. The photometer software is used to calculate the extinction change per minute (ΔE/min). The negative control values are subtracted.
14. To calculate the AraDH volume activity, following formula is used:
Volume activity [U/ml]= ΔE*min-1*ε-1*d-1*D
Enzyme activity (µmol*min-1) 1 U = 16.67 nkat
Change in extinction at 340 nm (min-1)
Extinction coefficient of NADPH at 340 nm (6.220 mM-1*cm-1)
thickness of the cuvette [cm] (300 μl in a 96 well MTP: 0.95 cm)
Dilution of the sample
15. For quantification of protein concentration, the protein assay PierceTM BCA Assay Kit is used. Suitable dilutions are measured according to the manufacturers instruction, adapted to the 96 well MTP. To generate a standard curve, that enables calculation of the sample protein concentration, bovine serum albumin (BSA) is used with concentrations from 0 mg/ml to 2 mg/ml. For the measurement, 20 µl of the sample dilutions as well as BSA dilutions are mixed with each 200 µl of pre mixed solution A+B (50:1). The solutions are incubated for at least 40 minutes at 37°C. The blue color, resulting from the biuret-reaction can be quantified photometrically at 562 nm.
16. The final step is the calculation of the specific enzyme activity in the SI unit [nkat/mg] that is done by dividing the volume activity [nkat/ml] with the protein concentration [mg/ml].