Mdh (BBa_K1585200) is our best Basic Part because it is a complete new sequence for a new catalytic sythesis that was not distributed at the registry before and is E. coli codon optimized.
A special coding sequence for an enzyme that catalyzes methanol to formaldehyde and is characterized and confirmed in its fuction, deserves to be honored with the best new Basic Part Award.
The characterization of the coding sequence was done with a RBS (B0034) in a T7 driven expression backbone. The expression of this coding sequence is guaranteed when B0034.Mdh is used.
To test if the expressed Mdh is functional, we modified a colorimetric and fluorescent formaldehyde assay described by T. Nash. In the in the presence of formaldehyde the yellow and fluorescing diacetyl-dihydro lutidine is formed. In the reaction catalyzed by the Mdh methanol is converted to formaldehyde which can be detected by the mentioned assay.
First, the different cells as well as their respective fragments and lysate supernatants were screened for formaldehyde production. The samples were taken 6 h after induction from shake flask cultures. Several cells, fragments and supernatants showed a production of formaldehyde but the Mdh activity in the intact cells was higher for each strain.
The formaldehyde assay was repeated only with whole cells and additional samples taken 20h after induction. Moreover, the assay was conducted not only at 37 °C but also at 30 °C. The GlgC expressing BL21 Gold was used as a negative control. Again, in several strains functional Mdh could be detected and some general conclusions could be drawn:
- More formaldehyde was produced in the samples taken 6 h after induction
- Strains cultivated at 37 °C show a stronger response than the same ones cultivated at 30 °C
- The assay works better and faster at an incubation temperature of 37 °C
By far the highest formaldehyde production was observed in the BL21 Gold (DE3) cells 6 h after induction cultivated at 37 °C despite its formation of inclusion bodies.
To show the significance of the production of formaldehyde the assay was conducted with the Mdh expressing BL21 Gold (DE3) cultivated in the labled methanol experiment along with the BL21 Gold (DE3) expressing GlgC as a negative control, both in multiple replicates.
The Mdh expressing strain showed significantly more formaldehyde production indicating a functional expression of the Mdh.