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Latest revision as of 03:59, 6 October 2011

Project Digestion

1. Introduction

Insect bodies are covered with hard integument mainly composed of chitin. To decompose the integument, we used ChiA gene, which encodes secreted chitinase. In order to measure the chitinase activity of the culture supernatant, we evaluated the effects of the medium and cell growth.

ChiA gene we used is derived from Streptomyces avermitilis. S. avermitilis is a kind of prokaryotic bacteria and they produce chitinase.[2][3] We extracted this gene from S. avermitilis and introduced it into Escherichia coli. This gene also encodes a secretion-signal sequence so that the chitinase will be secreted without the cell lysis of the E.coli.

For characterizing the chitinase activity, we used the DNS assay, which is the method to determine the quantity of reducing sugar. In our assay, we determine the quantity of N-acetylglucosamine in a culture supernatant. Since the other materials in the supernatant can react with DNS, it was needed to examine if this assay is available for the quantitative characterization of chitinase activity. We also made a model to assess the enzyme activity in the supernatant from the pre-experiment.

2. Method

Construction

We designed the following construction to enable the secretion of the chitinase, ChiA1. This gene is regulated by a strong lactose promoter, BBa_R0011. We used Streptmyces’s RBS into this construction, because in reference article [1] that RBS is used to allow E.coli to secrete the protein.

Assay

We performed 3,5-Dinitrosalicylic acid assay (DNS assay), because this assay takes less time, costs a less money and is used in the previous article measuring chitinase activities [1]. DNS assay is based on this fact: Being boiled, 3,5-dinitorosalicylic acid (DNS), whose color is yellow, reacts with reducing saccharide by boiling and changes into 3-amino-5-nitorosalicylic acid, whose color is brawn. [4][5]

The more the amount of reducing sugar is, the more this coloring reaction proceeds. We can quantitatively evaluate the degree of this coloring reaction by measuring OD 550, because OD 550 is indirectly the amount of the reducing sugar.

We will react DNS reagent with the media where chitin and E.coli introduced chitinase gene are added. If chitinase is secreted in media, chitin is decomposed into reducing sugar, for example, N-acetylglucosamin and coloring reaction proceeds. Therefore, by measuring OD 550, we can indirectly measure chitinase activities.

3. Result

This figure shows the overall of DNS assay The detail of these experiment was written in Protocol

To measure chitinase activity by DNS assay, we thought to need four following things to get accurate result. We need

to plot the relationship of reducing sugar concentration and OD 550
to evaluate the impact of components of media on OD 550
to evaluate the impact of remeineded E.coli in media on OD 550
to measure chitinase activity

3-1. Standard Measurement for ChiA1.

In order to know the relationship between OD550 and the amount of reducing sugar, we prepared the standard curve by using glucose as standard. It is known that the correlation between glucose concentration and the absorbance can be plotted in a linaer way.
From the result, a strong correlation between glucose concentration and its OD550 was observed.

Fig.1: Absorbance550 vs. glucose concentration. r²=0.98936.

**3-2. Consideration of medium and growth of E.coli.**

To get the accurate values of absorbance, it is needed to consider the residual E.coli in the supernatant. While the supernatant is incubated for several hours for the enzyme reaction, the cells can grow, produce chitinase and consume reducing sugar for their metabolism, which should influence to the result of DNS assay.

3-2-1 The time-course change of OD 550 of the supernatant.

In this experiment, we checked if the cells were still observed in the supernatant and assayed the growth behavior of cells. Figure 2 shows the time-course change of OD 550 of supernatant. The absorbance values of M9 were hardly increased and they were smaller than 0.2 during the incubation. The absorbance values of SOC were also increased slowly, but were higher than those of M9. The absorbance of values of Plusgrow Ⅱ were increased obviously, especially between the 3h and 5h, and much higher than those of M9 and SOC.

From figure 2, the increase in absorbance of Plusgrow Ⅱ was thought to be due to the cell growth. So, we considered that the cells also existed in M9 and SOC medium though the significant cell growth couldn't observed.

Fig.2: Absorbance of supernatant itself.

3-2-2 The DNS assay of the supernatant.

In this experiment, we evaluated the influence of the cells and its growth to the DNS assay. In other words, we confirmed if cells consume reducing sugar and affect the result of DNS assay. Figure 3 shows the absorbance of each culture supernatant. The values of M9 and Plusgrow Ⅱ were almost zero during the incubation. The absorbance of SOC was observed, but there was little change during the incubation.

From figure 2 and 3, the cell growth didn't affect to the change of absorbance. About M9 and SOC medium, each medium contains only glucose as sugar source and it is considered that glucose was mostly consumed after overnight culture. This is the reason why the cell growth didn't observed. On the other hands, Plusgrow Ⅱ didn't contain reducing sugar, but the cell growth was observed. It can be considered that even if Plusgrow Ⅱ doesn't contain any reducing sugar, the cells consume polysaccharide for their metabolism.

Fig.3: The influence of each culture supernatant to the DNS assay with time. Each supernatant was diluted 25 fold with water. Data points and error bars correspond to the mean and the standard deviation of three time experiments.

3-2-3 The DNS assay of medium.

We checked the influence of each medium to the DNS assay. Figure 4 shows the background absorbance of each medium. The absorbance of M9 was 1.7±0.1, SOC was1.227±0.007, and Plusgrow Ⅱ was 0.17±0.02.

The data of Plusgrow Ⅱ supports the fact that little reducing sugar is contained in this medium.

Fig.4: The influence of each medium to the DNS assay. Each medium was diluted 25 fold with water. Data points and error bars correspond to the mean and the standard deviation of three time experiments.

4. Discussion

Plusgrow Ⅱ should be suit for the DNS assay in the first three hours.

From three experiments: 3-2-1, 2, and 3, Plusgrow Ⅱ is good for characterizing the chitinase activity using the DNS assay in the first three hours. Firstly, cell number didn't increase so much in the first three hours. So, we can perform the chitinase assay that based on the premise that the enzyme concentration is nearly constant. Secondly, since E.coli can grow in the supernatant without having the influence to the result of DNS assay, the residual E.coli doesn't consume reducing sugar, and N-acetylglucosamine will be assayed quantitatively by DNS.

5.Achievment

We aimed at creating prefect chitinase constraction. However, We don't get this because of time shortage.
Finally, we could creat following constraction.

Moreover, We succesed modeling of chitinase activity. Modeling

Reference

[1] “Actinobacteria.” Internet: http://en.wikipedia.org/wiki/Actinobacteria [Nov. 5, 2011]

[2] H. Ikeda, J. Ishikawa, A. Hanamoto, M. Shinose, H. Kikuchi, T. Shiba, Y. Sakaki, M. Hattori, S. Omura, “Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis.” Nat Biotechnol., vol. 21, no. 5 pp. 526-31, Apr. 2003

[3] S. Omura, H. Ikeda, J. Ishikawa, A. Hanamoto, C. Takahashi, M. Shinose, Y. Takahashi, H. Horikawa, H. Nakazawa, T. Osonoe, H. Kikuchi, T. Shiba, Y. Sakaki, M. Hattori, “Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.” Proc Natl Acad Sci U S A. vol. 98, no. 21 pp. 12215-20, Oct. 9

[4] H. Sakuzou, ”還元糖の定量法(生物化学実験法)(kangento no teiryoho)” Kyoto University: Japan Scientific Soceties Press

[5] S. Kongruang, M. J. Han, C. I. Breton, M. H. Penner, “Quantitative Analysis of Cellulose-Reducing Ends.” Appl Biochem Biotechnol. Vol. 113, no. 116 pp. 213-31, Spring 2004

@@ Line 4: / Line 4: @@
 = '''Project Digestion''' =
-== '''Introduction''' ==
+== '''1. Introduction''' ==
+Insect bodies are covered with hard integument mainly composed of chitin. To decompose the integument, we used ChiA gene, which encodes secreted chitinase. In order to measure the chitinase activity of the culture supernatant, we evaluated the effects of the medium and cell growth.<br>
-Streptomyces is a kind of prokaryotic bacteria which decompose bodies in nature[1]. We extract chitinase gene from this bacterium and introduce into Escherichia coli. Secretion-signal sequences are included in this gene so that the protein coded by them will go out without occurring cell lysis. After assembling all genes, we examined the activity of this enzyme in quantitative way.
-=='''Method'''==
+ChiA gene we used is derived from ''Streptomyces avermitilis''. ''S. avermitilis'' is a kind of prokaryotic bacteria and they produce chitinase.[2][3] We extracted this gene from ''S. avermitilis'' and introduced it into ''Escherichia coli''. This gene also encodes a secretion-signal sequence so that the chitinase will be secreted without the cell lysis of the ''E.coli''.<br>
-==='''Construction'''===
-We created following construction to allow secretion of chitinase, chiA1. This gene is regulated by lactose promoter, BBa_R0011. We used Streptmyces’s RBS into this constructions, because reference article [1] used that to allow E.coli to secrete the protein.<br/>
-[[File:キャプチャ.PNG]]<br/>
-==='''Assay'''===
+For characterizing the chitinase activity, we used the DNS assay, which is the method to determine the quantity of reducing sugar. In our assay, we determine the quantity of N-acetylglucosamine in a culture supernatant. Since the other materials in the supernatant can react with DNS, it was needed to examine if this assay is available for the quantitative characterization of chitinase activity. We also made a model to assess the enzyme activity in the supernatant from the pre-experiment.<br>
-We performed the quantitative assay, 3,5-Dinitrosalicylic acid assay (DNS method). This assay takes a little time, costs a little money and was used previous article [1]. DNS assay is based on this fact: 3,5-dinitorosalicylic acid (DNS), whose color is yellow, reacts with reducing saccharide by boiling and changes into 3-amino-5-nitorosalicylic acid, whose color is brawn. We can measure the degree of the coloring reaction by OD 550<br>
+=='''2. Method'''==
+==='''Construction'''===
+We designed the following construction to enable the secretion of the chitinase, ChiA1. This gene is regulated by a strong lactose promoter, BBa_R0011. We used ''Streptmyces''’s RBS into this construction, because in reference article [1] that RBS is used to allow ''E.coli'' to secrete the protein.<br/>
+[[File:Digestion_Constrctin1.png]]<br/>
-This coloring reaction
+==='''Assay'''===
+We performed 3,5-Dinitrosalicylic acid assay (DNS assay), because this assay takes less time, costs a less money and is used in the previous article measuring chitinase activities [1]. DNS assay is based on this fact: Being boiled, 3,5-dinitorosalicylic acid (DNS), whose color is yellow, reacts with reducing saccharide by boiling and changes into 3-amino-5-nitorosalicylic acid, whose color is brawn. [4][5]<br>
-Therefore, the more the amount of  is
+The more the amount of reducing sugar is, the more this coloring reaction proceeds. We can quantitatively evaluate the degree of this coloring reaction by measuring OD 550, because OD 550 is indirectly the amount of the reducing sugar.
+We will react DNS reagent with the media where chitin and E.coli introduced chitinase gene are added. If chitinase is secreted in media, chitin is decomposed into reducing sugar, for example, N-acetylglucosamin and coloring reaction proceeds. Therefore, by measuring OD 550, we can indirectly measure chitinase activities. <br>
-If chitinase is secreted in media by E.coli, chitin added in the media will be decomposed into reducing sugar, for example, N-acetylglucosamin. Therefore, the absorbance of the media added DNS will increase. <br>
 [[File:Kyoto-digestion-DNSassay1.jpg]]<br/>
+=='''3. Result'''==
-Therefore, the absorbance of the liquid increases in direct proportion to the amount of reducing sugar.
+This figure shows the overall of DNS assay
+[[File:キチナーゼ.PNG]]
+The detail of these experiment was written in <html><a href="/Team:Kyoto/Measurement#Measurement">Protocol</a></html>
-==='''Procedure'''===
-To carry out DNS assay, we thought four following dates would be required. These are
-#the relationsip to reducing sugar concntration and absorbance 550
-#the assesment of the component in media which can effect absobance 550
-#the assesment of effect of E.coli in media
-#the effect of chitinase
-There are still two concerns, affection of medium itself and remained E.coli interruption. Perhaps, medium supernatant contains some compounds reducing 3,5-dinitorosalicylic acid and E.coli which remain there even after centrifusion. These effects can cause error in the value of absorbance. Reducing substance can react with 3,5-dinitorosalicylic acid and change the color of the supernatant. E.coli may consume reducing sugars derived from chitin and prevent 3,5-dinitorosalicylic acid from reducing reaction. In order to estimate the extents of these tow effects and build the most appropriate experiment system, we conduct these following assays.
+To measure chitinase activity by DNS assay, we thought to need four following things to get accurate result. We need
+#to plot the relationship of reducing sugar concentration and OD 550
+#to evaluate the impact of components of media on OD 550
+#to evaluate the impact of remeineded E.coli in media on OD 550
+#to measure chitinase activity<br>
-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.
-===='''preliminary experiments'''====
-'''1-1:build a standard curve'''
+==='''3-1. Standard Measurement for ChiA1.'''===
+In order to know the relationship between OD550 and the amount of reducing sugar, we prepared the standard curve by using glucose as standard. It is known that the correlation between glucose concentration and the absorbance can be plotted in a linaer way.<br>
+From the result, a strong correlation between glucose concentration and its OD550 was observed.
+[[File:Kyoto-ChiA1Standard0925.png|thumb|center|350px|Fig.1: Absorbance550 vs. glucose concentration. ''r''<sup>''2''</sup>=0.98936.]]
-{|
+==='''3-2. Consideration of medium and growth of ''E.coli''.'''===
-|Sample
-  |  glucose solution(various concentration from 0.20 mM to 1.60 mM)
-|-
-|Blank
-  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
-|}
+To get the accurate values of absorbance, it is needed to consider the residual ''E.coli'' in the supernatant. While the supernatant is incubated for several hours for the enzyme reaction, the cells can grow, produce chitinase and consume reducing sugar for their metabolism, which should influence to the result of DNS assay.<br><br>
-#added 240㎕ DNS reagent to 80㎕sample
+:'''3-2-1 The time-course change of OD 550 of the supernatant.'''
-#heated it for 5 min in boiling water and then cooled it in water.
+:In this experiment, we checked if the cells were still observed in the supernatant and assayed the growth behavior of cells. Figure 2 shows the time-course change of OD 550 of supernatant. The absorbance values of M9 were hardly increased and they were smaller than 0.2 during the incubation. The absorbance values of SOC were also increased slowly, but were higher than those of M9. The absorbance of values of Plusgrow &#8545; were increased obviously, especially between the 3h and 5h, and much higher than those of M9 and SOC.<br><br>
-#added it distilled water by 2ｍｌ
+:From figure 2, the increase in absorbance of Plusgrow &#8545; was thought to be due to the cell growth. So, we considered that the cells also existed in M9 and SOC medium though the significant cell growth couldn't observed. <br>
-#measured absorbance in 550nm
-The standard curve is shown in fig 1.
+[[File:Kyoto-Absorbance_of_supernatant.png|thumb|center|400px|Fig.2: Absorbance of supernatant itself.]]<br>
+:'''3-2-2 The DNS assay of the supernatant.'''
+:In this experiment, we evaluated the influence of the cells and its growth to the DNS assay. In other words, we confirmed if cells consume reducing sugar and affect the result of DNS assay. Figure 3 shows the absorbance of each culture supernatant. The values of M9 and Plusgrow &#8545; were almost zero during the incubation. The absorbance of SOC was observed, but there was little change during the incubation.<br><br>
+:From figure 2 and 3, the cell growth didn't affect to the change of absorbance. About M9 and SOC medium, each medium contains only glucose as sugar source and it is considered that glucose was mostly consumed after overnight culture. This is the reason why the cell growth didn't observed. On the other hands, Plusgrow &#8545; didn't contain reducing sugar, but the cell growth was observed. It can be considered that even if Plusgrow &#8545; doesn't contain any reducing sugar, the cells consume polysaccharide for their metabolism.<br><br>
-'''1-2:evaluate the affection of medium'''
+[[File:Kyoto-DNS_assay_of_supernatant.png|thumb|center|400px|Fig.3: The influence of each culture supernatant to the DNS assay with time. Each supernatant was diluted 25 fold with water. Data points and error bars correspond to the mean and the standard deviation of three time experiments.]]<br>
-we examined the interruption based on the components of media(SOC, plas-grow and M9)
-{|
-|Sample
-  |  each medium
-|-
-|Blank
-  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
-|}
-#added 240㎕ DNS reagent to 80㎕sample
-#heated it for 5 min in boiling water and then cooled it in water.
-#added it distilled water by 2ｍｌ
-#measured absorbance in 550nm
-The result is shown in fig 2.
-'''1-3:measurement the effect of remainded E.coli'''
-{|
-|Sample
-  |  each medium
-|-
-|Blank
-  |  mixed liquid (240μℓDNS reagent plus 1760㎕distilled water　)
-|}
-This assay was performed three times for each medium
-#poured the medium　cultured E.coli overnight 1.2 ㎕ into each five microcentritube.
-#centrifuged them for 5 min at 5,000 rpm
-#prepared new five microcentritube and move 800㎕ the supernatants into each of them.
-#measure the OD550 of one tube（use fresh medium as a blank in following assays）
-#one hour after, we measured OD 550 of other tube
-#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.
-#added 240㎕ DNS reagent and heated it for 3 min in boiling water and then cooled it in water.
-#two, three, five hours after, we did above operation, taking supernatant, measured OD500, heating and cooling, applying DNS reagent and heating and cooling again.
-#added all sample tube (containing 320㎕ solution) distilled water by 2ｍｌ and measure the absorbance of them in 550nm.
-The results of measurement OD550 are shown in fig 3 and of measurement DNS assay are shown in fig 4.
-[[File:キチナーゼ.PNG]]
-=='''Result'''==
-. Standard Measurement for ChiA1.
-:From the result, a strong correlation between glucose concentration and its A<sub>550</sub> was observed.
-[[File:Kyoto-ChiA1Standard0925.png|thumb|center|350px|Fig.1: Absorbance550 vs. glucose concentration. ''r''<sup>''2''</sup>=0.98936.]]
-. Consideration of medium and growth of ''E.coli''.
-:We checked the influence of each medium to the DNS assay. Figure 2 shows the background absorbance of each medium. The absorbance of M9 was 1.7&plusmn;0.1, SOC was1.227&plusmn;0.007, and Plusgrow &#8545; was 0.17&plusmn;0.02.
-<div style="width:100%; float:left">
-[[File:Kyoto-DNSassayforeachmedium.png|thumb|left|250px|Fig.2: The influence of each medium to the DNS assay. Data points and error bars correspond to the mean and the standard deviation of three time experiments.]]
-[[File:Kyoto-DNSassayforeachmediumafterovernighculture.png|thumb|left|250px|Fig.3:]]
-[[File:Kyoto-cellpopulationafterovernightculture.png|thumb|left|250px|Fig.4:]]
-</div>
-=='''Discussion'''==
+:'''3-2-3 The DNS assay of medium.'''
-From result of preliminary experiments, we found several problems.
+:We checked the influence of each medium to the DNS assay. Figure 4 shows the background absorbance of each medium. The absorbance of M9 was 1.7&plusmn;0.1, SOC was1.227&plusmn;0.007, and Plusgrow &#8545; was 0.17&plusmn;0.02.<br><br>
-・the affection of medium itself
+:The data of Plusgrow &#8545; supports the fact that little reducing sugar is contained in this medium.
-The components of each medium also reduced 3,5-dinitorosalicylic acid and would
+[[File:Kyoto-DNSassayforeachmedium.png|thumb|center|400px|Fig.4: The influence of each medium to the DNS assay. Each medium was diluted 25 fold with water. Data points and error bars correspond to the mean and the standard deviation of three time experiments.]]
-cause error in the assay.
-・interruption of remaining E.coli.
+=='''4. Discussion'''==
-Even though we use the centrifugal supernatant, there was still some E.coli.
+==='''Plusgrow &#8545; should be suit for the DNS assay in the first three hours.'''===
-we found they could interrupt data because they would decompose reducing sugers.
+From three experiments: 3-2-1, 2, and 3, Plusgrow &#8545; is good for characterizing the chitinase activity using the DNS assay in the first three hours. Firstly, cell number didn't increase so much in the first three hours. So, we can perform the chitinase assay that based on the premise that the enzyme concentration is nearly constant. Secondly, since ''E.coli'' can grow in the supernatant without having the influence to the result of DNS assay, the residual ''E.coli'' doesn't consume reducing sugar, and N-acetylglucosamine will be assayed quantitatively by DNS.<br><br>
-To overcome these barriers, we decided detail plan of our assay.
+=='''5.Achievment'''==
+We aimed at creating prefect chitinase constraction. However, We don't get this because of time shortage.<br>
+Finally, we could creat following constraction.<br>
+[[File:Digestion_Constraction2.png]]
-From the result fig 3, SOC medium cultured E.coli overnight would still include too much amount of reducing materials and, from fig 3. plas-grow enabled reminded E.coli to grow rapidly.  However, as for M9 medium,
+Moreover, We succesed modeling of chitinase activity.
+<html><a href="/Team:Kyoto/Digestion/Modeling">Modeling</a></html><br>
 =='''Reference'''==
@@ Line 162: / Line 99: @@
 [3] S. Omura, H. Ikeda, J. Ishikawa, A. Hanamoto, C. Takahashi, M.  Shinose, Y. Takahashi, H. Horikawa, H. Nakazawa, T. Osonoe, H. Kikuchi, T. Shiba, Y. Sakaki, M. Hattori, “Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.” Proc Natl Acad Sci U S A. vol. 98, no. 21 pp. 12215-20, Oct. 9
-[4] H. Sakuzou, ”還元糖の定量法(生物化学実験法)” Kyoto University: Japan Scientific Soceties Press
+[4] H. Sakuzou, ”還元糖の定量法(生物化学実験法)(''kangento no teiryoho'')” Kyoto University: Japan Scientific Soceties Press
 [5] S. Kongruang, M. J. Han, C. I. Breton, M. H. Penner, “Quantitative Analysis of Cellulose-Reducing Ends.” Appl Biochem Biotechnol. Vol. 113, no. 116 pp. 213-31, Spring 2004

Team:Kyoto/Digestion