Part:BBa_K1585241
AP19.Mdh2.Hps.Phi.Xpk.B0015
polycistronic expression part for methanol assimilation genes
Usage and Biology
We successfully assembled all required genes in a polycistronic frame behind an AP19 promoter in a backbone with kanamycin resistance. This was confirmed through sequencing results.
A SDS gel showed that all four genes are expressed. Next, we tested the physiology and the enzyme activity of a strain that carries our construct.
Expression test of polycistronic plasmid on SDS-Page. The cell pellet of E. coli BL21 Gold (DE3) that carries the polycistronic construct downstream of Anderson Promoter 19 was applied on an SDS-PAGE.
Successful sequencing of polycistronic construct
Proof of Mdh Activity in Methanol Conversion Strain
As the Mdh represents the bottleneck of the whole MCC pathway we tested its activity in the strains with the polycistronic plasmid. For this the assay to detect formaldehyde first described by Nash was done with these strains. Whole cells, cell fragments and lysate supernatants were tested for the polycistronic strains. The highest formation of formaldehyde could be observed in the assay using whole cells.
Activity of the Mdh in strain harboring the polycistronic construct The strain with the polycistronic plasmid (#POLY#) shows significantly more Mdh activity than the negative control (#OHES#) expressing GlgC and the blank consisting of Nash reagent and buffer. Fluorescence measured at a gain of 75.
It was proven that the Mdh is expressed functionally in the strains containing the polycistronic construct. However, if compared to the activity of the strain only expressing the Mdh it is rather low (for comparison see data above).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 638
Illegal AgeI site found at 3893 - 1000COMPATIBLE WITH RFC[1000]
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