Team:Kyoto/Digestion/DNS method

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= '''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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== 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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Sample    media(with antibiotic)
 
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Blank     mixed liquid (240μℓDNS reagent plus 1760㎕distilled water )
 
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Reagent   DNS
 
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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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[[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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[[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
 

Latest revision as of 21:21, 4 October 2011