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Team:Osaka/Tests
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<h2>Tests</h2> | <h2>Tests</h2> | ||
| - | <h3> | + | <h3>Congo Red clearing assay</h3> |
| - | We constructed <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K392039">K392039</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K392041">K392041</a> as tests constructs, containing the <i>E. coli</i> PelB secretion tag and <i>C. fermi</i> endoglucanase CenA or exoglucanase Cex, respectively. These constructs tested for cellulose degradation activity by carboxymethylcellulose (CMC) clearing, whereupon transformed bacteria are spotted onto agar plates infused with CMC. Degradation of CMC results in a pale-colored halo appearing around the spotted colony. | + | As an initial screening for cellulase activity, we used the Congo Red assay.<br> |
| + | We constructed <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K392039">K392039</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K392041">K392041</a> as tests constructs, containing the <i>E. coli</i> PelB secretion tag and <i>C. fermi</i> endoglucanase CenA or exoglucanase Cex, respectively. These constructs tested for cellulose degradation activity by carboxymethylcellulose (CMC) clearing, whereupon transformed bacteria are spotted onto agar plates infused with CMC and Congo Red. Degradation of CMC results in a pale-colored halo appearing around the spotted colony. | ||
<table> | <table> | ||
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<h3>Future work</h3> | <h3>Future work</h3> | ||
<p>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.</p> | <p>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.</p> | ||
| + | |||
| + | <h3>References</h3> | ||
| + | <li></li> | ||
| + | <li></li> | ||
| + | <li></li> | ||
Revision as of 21:42, 27 October 2010
Tests
Congo Red clearing assay
As an initial screening 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 tested for cellulose degradation activity by carboxymethylcellulose (CMC) clearing, whereupon transformed bacteria are spotted onto agar plates infused with CMC and Congo Red. Degradation of CMC results in a pale-colored halo appearing around the spotted colony.
 |
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| CenA constructs. Clockwise from left: CenA only; CenA with PelB; negative control. |
Cex constructs. Clockwise from top: Cex with PelB; Cex only; negative control. |
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
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