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| | {{Kyoto_Background}} | | {{Kyoto_Background}} |
| | {{Kyoto_WikiDesign}} | | {{Kyoto_WikiDesign}} |
| - | = '''3,5-dinitorosalicylic acid assay(DNS method)''' =
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| - | This assay is based on this fact: 3,5-dinitorosalicylic acid (DNS) is changed into 3-amino- 5-nitorosalicylic acid by reducing saccharide in reaction solution and the absorbance of this liquid increase in direct proportion to the amount of reducing sugar.
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| - | [[File:Kyoto-digestion-DNSassay1.jpg]]<br/>
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| - | To carry out this assay, we will be required following three things, that is,
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| - | *the relationsip to reducing sugar concntration and absorbance 550
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| - | *the assesment of effect of E.coli in media
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| - | *the effect of chitinase
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| - | == '''Method''' ==
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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.
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| - |
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| - | == preliminary experiments ==
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| - | 1-1:evaluate the affection of medium
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| - | we examined the interruption based on the components of media(SOC, plas-grow and M9)
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| - |
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| - | {|
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| - | |Sample
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| - | |media(with antibiotic)
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| - | |-
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| - | |Blank
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| - | |mixed liquid (240μℓDNS reagent plus 1760㎕distilled water )
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| - | |-
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| - | |Reagent
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| - | |DNS
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| - | |}
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| - | #added 240㎕ DNS reagent to 80㎕sample
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| - | #heated it for 5 min in boiling water and then cooled it in water.
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| - | #added it distilled water by 2ｍｌ
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| - | #measured absorbance in 550nm
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| - | 1-2:measurement the effect of remainded E.coli
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| - | This assay was performed three times for each medium
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| - | #poured the medium cultured E.coli overnight 1.2 ㎕ into each five microcentritube.
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| - | #centrifuged them for 5 min at 5,000 rpm
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| - | #prepared new five microcentritube and move 800㎕ the supernatants into each of them.
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| - | #measure the OD550 of one tube（use fresh medium as a blank in following assays）
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| - | #one hour after, we measured OD 550 of other tube
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| - | #take 80㎕ supernatant and move it into new tube and then heated it for 3 min in boiling water and then cooled it in water.
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| - | #added 240㎕ DNS reagent and heated it for 3 min in boiling water and then cooled it in water.
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| - | #two, three, five hours after, we did above operation, taking supernatant, measured OD500, heating and cooling, applying DNS reagent and heating and cooling again.
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| - | #added all sample tube (containing 320㎕ solution) distilled water by 2ｍｌ and measure the absorbance of them in 550nm.
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| - | == '''Result''''' ==
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| - | 1. Standard Measurement for ChiA1.
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| - |
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| - | [[File:Kyoto-ChiA1Standard0925.png|thumb|center|400px|Fig.1: Absorbance550 vs. glucose concentration. ''r''<sup>''2''</sup>=0.98936.]]
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| - | :From the result, a strong correlation between glucose concentration and its A<sub>550</sub> was observed.
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| - | 2. Consideration of medium and growth of ''E.coli''.
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| - | [[File:Kyoto-DNSassayforeachmedium.png|left|thumb|350px|Fig.2:]]
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| - | [[File:Kyoto-DNSassayforeachmediumafterovernighculture.png|thumb|center|350px|Fig.3:]]
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| - |
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| - | [[File:Kyoto-cellpopulationafterovernightculture.png|thumb|center|350px|Fig.4:]]
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| - | == '''Discussion''' ==
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| - | == '''Reference''' ==
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| - | [1]Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis
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| - | [2]Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites
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| - | [3]還元糖の定量法 (生物化学実験法)福井 作蔵
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| - | [4]Quantitative Analysis of Cellulose-Reducing Ends
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