Team:Osaka/Tests

Tests

Congo Red clearing assay

As an initial screen for cellulase activity, we used the Congo Red assay.
We constructed K392039 and K392041 as tests constructs, containing the E. coli PelB secretion tag and C. fermi endoglucanase CenA or exoglucanase Cex, respectively. These constructs were transformed into bacteria and the transformants spotted onto agar plates infused with carboxymethylcellulose (CMC) and Congo Red. Degradation of CMC results in a pale-colored halo appearing around the spotted colony.

CenA constructs. Clockwise from left: CenA only; CenA with PelB; negative control.	Cex constructs. Clockwise from top: Cex with PelB; Cex only; negative control.

From the results it appears that spots corresponding to secretion-tagged cellulase parts are brighter than non-tagged, suggesting higher CMC-clearing (ie. extracellular cellulose degradation).

Microscope observation of cell surface display

Since confirmation of surface display by antibody staining is too costly we decided to use fluorescent microscopy.
We made a test construct consisting of surface display tag attached to GFP, K392038. Yeast cells were transformed with this part and then stained with propidium iodine (PI), which binds to the cell walls but does not permeate the cell interior. The stained transformants then observed under a fluorescent microscope.
surface : Propidium Iodide
scale bar : 1 micrometer

As shown above, GFP intensity is co-localized with PI. Since PI is localized to the cell membrane, ie. exterior of the cell, we can infer that GFP is being displayed extracellularly. Some GFP can be seen dispersed around the cell; this might be Aga2-tagged GFP that has failed to bind to Aga1 on the cell surface.
We conclude that the Aga2 cell surface tag received from NYU works; now it only remains to test the effect of the display on cellulase activity, by constructing the appropriate test device(s).

Cellulase quantitative activity assay: DNS method

Future work

We created many parts (yeast ENO2 promoter, ADH1 terminator, SUC2 secretion signal etc as well as various cellulases such as xylanase and mannase) but did not have time to evaluate them. Also, devices containing 2 or more cellulases should have been constructed and assayed.

References