Part:BBa_K1585200:Experience

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Applications of BBa_K1585200

Single expression of Mdh

Mdh, Hps and Phi expressed in a pSB1A30 backbone on SDS page after IPTG induction with a RFP expression strain as negative control. All genes expressed in BL21 Gold (DE3), samples taken from 50 ml main culture in shake flasks after 6 h and 21 h after IPTG induction.

Expression test of polycistronic plasmid on SDS-Page

The cell pellet of E. coli BL21 Gold (DE3) that carries the polycistronic construct of the Methanol Condensation Cycle (MCC) downstream of Anderson Promoter 19 was applied on an SDS-PAGE.

Expression verification & localization of the Mdh

The expression of the Mdh behind a T7 promoter was verified by doing an SDS-PAGE. To localize the Mdh not only whole cells were used but also the fragments of lysed cells and the respective supernatant. The expected bands were clearly visible in all samples proving the expression of the Mdh. However, the Mdh specific band had the highest intensity in the sample of the cell fragments whereas the same band was only of slight intensity in the supernatant. This indicates that the Mdh is incorporated into inclusion bodies.

Expression test of #IGEM# An SDS-PAGE of the whole cell, the cell fragments and the lysate supernatant of the Mdh expressing BL21 Gold (#IGEM#) and the GlgC expressing BL21 Gold (#OHES#) as a negative control was run. The expected band at the weigth of the Mdh can be clearly seen in all #IGEM# samples. The according band with the strongest intensity occurs in the lane with #IGEM# fragments indicating the formation of inclusion bodies containg the Mdh.

To avoid the incorporation of the Mdh into inclusion bodies, the alternative E. coli strains SHuffle T7 Express and C43 were used. SHuffle T7 Express allows more efficient protein folding in the cytoplasm and lacks proteases whereas C43 allows the expression of toxic proteins. Additionally, cultivation at a lower temperature of 30°C was tested. It was shown that all strains were able to grow on M9 medium, however, another test for the expression and localization of the Mdh did not reveal major differences between the strains. In every case the Mdh was still incorporated into inclusion bodies.

Functionality of the expressed Mdh

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 6h 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 6h 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

Comparison of different strains at varying cultivation conditions The highest activity could be shown in BL21 Gold (DE3) at a cultivation temperature of 37 °C in M9 in a sample taken 6 h after induction.

By far the highest formaldehyde production was observed in the BL21 Gold (DE3) cells 6h 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 (n>35).

Formaldehyde formation during in vivo assay #IGEM# expresses Mdh and #OHES# expresses glgC as a negative control (4.5 h after induction). Both constructs were incorporated in vector pSB1A30 and the strains were cultivated at 37 °C. Each value is made from at least 35 replicates of each construct.

The Mdh expressing strain showed significantly more formaldehyde production indicating a functional expression of the Mdh.

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