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

It is test for surface representational signal ( from NY team →). We observed GFP with this signal.As shown below, GFP is co-localize with surface.Although cell line, Saccharomyces cerevisiae, is small, there was lower signals of GFP at the center of cell. For above reasons, the signal worked obviously.
surface : Propidium Iodide
scale bar : 1 micrometer

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