Team:Osaka/Tests

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<h3>Congo Red clearing assay</h3>
<h3>Congo Red clearing assay</h3>
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As an initial screen for cellulase activity, we used the Congo Red assay.<br>
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<p>
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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 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.
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As an initial screen for cellulase activity, we used the Congo Red assay. Congo Red is the sodium salt of benzidinediazo-bis-1-naphthylamine-4-sulfonic acid and binds strongly with amyoids such as cellulose yielding a red colloidal solution.  When the cellulose is degraded the red color disappears leaving a pale-colored halo.<br>
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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 were transformed into bacteria and the transformants spotted onto agar plates infused with carboxymethylcellulose (CMC) bound with Congo Red. Degradation of CMC results in a halo appearing around the spotted colony.
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<table>
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<table padding="10">
<tr>
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<td><img src="https://static.igem.org/mediawiki/2010/6/61/Cmc1.png" width="200"></td>
<td><img src="https://static.igem.org/mediawiki/2010/6/61/Cmc1.png" width="200"></td>
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</table>
</table>
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).<br>
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).<br>
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</p>
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<h3>Microscope observation of cell surface display</h3>
<h3>Microscope observation of cell surface display</h3>
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<p>It is test for surface representational signal ( from NY team <a href="https://2010.igem.org/Team:Osaka/Collaborations">→</a>). We observed GFP with this signal.As shown below, GFP is co-localize with surface.Although cell line, <i>Saccharomyces cerevisiae</i>, is small, there was lower signals of GFP at the center of cell. For above reasons, the signal worked obviously.
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<p>Since confirmation of surface display by antibody staining is too costly we decided to observe GFP-chimera protein.<br>
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<br>surface : Propidium Iodide</br>
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We made a test construct consisting of surface display tag attached to GFP, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K392038">K392038</a>. Yeast cells were transformed with this part and then stained with propidium iodine (PI), which stein live cell's wall, because PI cannnot transmit through cell membrane. Therefore we stained cell with PI before fixation. The stained transformants were then observed under a fluorescent microscope.<br>
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scale bar : 1 micrometer</p>
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<img src="https://static.igem.org/mediawiki/2010/5/5a/MS1.png" width="350"><br>
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<img src="https://static.igem.org/mediawiki/2010/5/5a/MS1.png" width="350">
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scale bar : 1 micrometer<br>
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<br></br>
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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.<br>
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<h3>Cellulase quantitative activity assay: DNS method</h3>
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We conclude that the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K416004">Aga2 cell surface tag</a> 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).
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</p>
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<h3>Cellulase quantitative assay by reducing sugar measurement</h3>
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<p>In basic solution, 3,5-dinitro salicylic acid, DNS, is reduced by reducing sugar.  We used this reaction to determinate the quantity of reducing sugar present in the medium which resulted from degradation of cellulose.<br>
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We characterized the <i>C. fermi</i> endoglucanase A part <a href="http://partsregistry.org/Part:BBa_K118023">BBa_K118023</a> as part of a device including lac promoter, RBS and terminator. Transformants were incubated in liquid medium for ~10h, then this cell culture was added to solutions containing cellulose, CMC or ASC respectively. After reaction, DNS was added and absorbance measured to quantify the amount of sugar present versus a calibration curve.
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<img src="https://static.igem.org/mediawiki/2010/f/f0/DNS_chart.png" width="800">
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</p>
<h3>Future work</h3>
<h3>Future work</h3>
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<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>  
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<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>
<h3>References</h3>
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<li></li>
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<ul>
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<li></li>
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<li>Production and Applications of Cellulase Laboratory Procedures Handbook, Mary Mandels (1974)</li>
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<li></li>
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<li>Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate diffusion assay, John Carder (1985)</li>
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</ul>
</div>
</div>

Latest revision as of 03:15, 28 October 2010

Tests

Congo Red clearing assay

As an initial screen for cellulase activity, we used the Congo Red assay. Congo Red is the sodium salt of benzidinediazo-bis-1-naphthylamine-4-sulfonic acid and binds strongly with amyoids such as cellulose yielding a red colloidal solution. When the cellulose is degraded the red color disappears leaving a pale-colored halo.
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) bound with Congo Red. Degradation of CMC results in a 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 observe GFP-chimera protein.
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 stein live cell's wall, because PI cannnot transmit through cell membrane. Therefore we stained cell with PI before fixation. The stained transformants were then observed under a fluorescent microscope.

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 assay by reducing sugar measurement

In basic solution, 3,5-dinitro salicylic acid, DNS, is reduced by reducing sugar. We used this reaction to determinate the quantity of reducing sugar present in the medium which resulted from degradation of cellulose.
We characterized the C. fermi endoglucanase A part BBa_K118023 as part of a device including lac promoter, RBS and terminator. Transformants were incubated in liquid medium for ~10h, then this cell culture was added to solutions containing cellulose, CMC or ASC respectively. After reaction, DNS was added and absorbance measured to quantify the amount of sugar present versus a calibration curve.

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

  • Production and Applications of Cellulase Laboratory Procedures Handbook, Mary Mandels (1974)
  • Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate diffusion assay, John Carder (1985)
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