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| - | ===Procedure===
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| - | DNS method
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| - | We examined the quantitative relation between absorbance and the volume of sugar and then expressed it onto a straight line graph (result fig 1:リンク).
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| - | :We led E.coli introduced chitinase gene had secreted disassemble chitin into N-acetylglucosamine and other sugar derivatives in water.
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| - | :After passing enough time (_min), we added this liquid 0.2 ml into DNS reagent 0.6 ml (<html><a href="https://2011.igem.org/Team:Kyoto/Protocol">How to prepare</a></html>) and boiled this solution for 5 min. After cooling this soluion, We diluted with water to 5 ml and assay the absorbance in 550 nm.
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| - | ::The results of this measurement and the fig 1 graph enabled us to calculate the amount of digested chitin, showing the relative activity of chitinase. We did same examination about commercial chitinase derived from Streptomyces and exhibited in result fig 2:リンク
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| - | :: '''２－１：Evaluate the accidental change of absorbance'''
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| - | ::Even though we centrifuge media to remove E,coli, there might be still some E.coli in solution.For this reason, we examined accidental errors in absorbance as the following way before conducting DNS assay. The speed of increasing the number of E.coli would depend on media so that we examined all kind of media which we could have used in DNS assay.
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| - | ::・cultured E.coli overnight in LB medium<br>
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| - | ::・poured the medium (above 1㎕) into each three microcentritube and measured OD<br>
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| - | ::Following operations were conducted to all tubes.<br>
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| - | ::・centrifuge for 5 min at 5,000 rpm and move 1㎕ the supernatant to new microcentritube. (It was ::incubated at 37℃ until this experiment was finished.)<br>
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| - | ::・measured absorbance <br>
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| - | ::・one hour after, we measured absorbance and took 200㎕ the solution to new tube and heated it ::for 3 min in boiling water and then cooled it in water.<br>
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| - | ::・applied 200㎕ DNS reagent and heated it for 5 min in boiling water and then cooled it in water.<br>
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| - | ::・two, three, five hours after, we did above operation, taking supernatant, measured absorbance,heating and cooling, applying DNS reagent and heating and cooling again.<br>
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