Part:BBa_K3819077
EGL7 plasmid
The cellulase plasmid expresses cellulase with Type1 dockerin (derived from clostridium fibrogiformis) that can be secreted out of the cell. The cellulase and dockerin are connected by a flexible linker (GGGGS)3. Type1 dockerin derived from Clostridium ophagium Type1 cohesion could be bonded to the skeleton derived from Clostridium ophagium Type1 cohesion, thus anchoring cellulase on the skeleton. There were two types of dockerin and cohesion, namely Type1 and Type2. Type1 Dockerin derived from the same species could bond with the cohesion of that species, but Type1 dockerin could not bond with the Cohesion of Type2.
Usually, Type1 Dockerin and Cohesion were used for the bonding between enzymes and skeleton, while Type2 Dockerin and Cohesion were used for the bonding between skeleton and anchor scaffold. In addition, there should be a tag on the cellulase plasmid, and the short peptide expressed by the tag can bind with the first antibody and the second antibody with a fluorescent dye, so as to verify the binding of cellulase and skeleton in the future.
Finally, we successfully expressed this recombinant plasmid and detected the activity of cellulase EGL7, which means EGL7 can work normally. And the results are as follows.
- Figure 1. Dot Blot analysis of EGL7. The sample was gained after fermentation in YPD for 96h. EGL7(His-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.
- Figure 2. Enzyme activity detection of EGL7. The solution was gained after 96h of fermentation. 2ml fermentation supernatant reacted with 2ml 1% Xylan solution for 30min, then added 5ml DNS to shut down the process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while the control just showed unobvious color change.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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