Team:Kyoto/Digestion

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(Project Digestion)
 
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{{Kyoto_Background}}
{{Kyoto_Background}}
{{Kyoto_WikiDesign}}
{{Kyoto_WikiDesign}}
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<div id="main">
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= '''Project Digestion''' =
= '''Project Digestion''' =
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== '''Introduction''' ==
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== '''1. Introduction''' ==
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Streptomyces is a kind of prokaryotic bacteria which decompose bodies in nature. We extract protease and chitinase genes from this bacterium and introduce into Escherichia coli. Secretion-signal sequences are included in these genes so that the proteins coded by them will go out without occurring cell lysis.  After assembling all genes, we examined the activity of these two enzymes in both of qualitative and quantitative ways.
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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>
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== '''Method''' ==
 
-
===Construction===
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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>
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We created following constructions to allow secretion of Serine protease, SAM-P20 and chitinase, chiA1. These genes are regulated by lactose promoter, BBa_R0011. We used Streptmyces’s RBS into these constructions, because reference article [1] used them to allow E.coli to secrete these proteins.
 
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[[File:Kyoto-digestion-costruction1.jpg]]<br/>
+
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>
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[[File:Kyoto-digestion-costruction2.jpg]]<br/>
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===Assay===
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=='''2. Method'''==
 +
==='''Construction'''===
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Serine Protease
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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/>
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:Experiment 1 : Skim milk-hydroryzing assay
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[[File:Digestion_Constrctin1.png]]<br/>
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:In order to identify the expression of SAM-P20 gene, a skim milk-hydroryzing assay was performed. A plate containing 2% skim milk, IPTG(final concentration, 0.5mM), and 0.1% yeast extract was used for this assay.
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 +
==='''Assay'''===
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:Experiment 2 : Measurement of enzyme activity
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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>
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:In order to measure the activity of serine protease, the fluorescent assay was done, using fluorescein-labeled casein as a substrate.
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Chitinase A
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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.
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:Experiment 1
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:Experiment 2 : 3,5-Dinitrosalicylic 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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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>
[[File:Kyoto-digestion-DNSassay1.jpg]]<br/>
[[File:Kyoto-digestion-DNSassay1.jpg]]<br/>
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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). We led chitinase E.coli had ::secreted disassemble chitin into N-acetylglucosamine and other sugar derivatives in water.  ::After passing enough time (_min), we added this liquid 1 ml into DNS reagent 3 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 25 ml and assay the absorbance in 550 nm.  
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=='''3. Result'''==
 +
 
 +
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>
 +
 
 +
 
 +
 
 +
 
 +
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>
 +
 
 +
 
 +
 
 +
 
 +
==='''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''.'''===
 +
 
 +
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>
 +
 
 +
:'''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 &#8545; were increased obviously, especially between the 3h and 5h, and much higher than those of M9 and SOC.<br><br>
 +
: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>
 +
 
 +
[[File:Kyoto-Absorbance_of_supernatant.png|thumb|center|400px|Fig.2: Absorbance of supernatant itself.]]<br>
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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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:'''3-2-2 The DNS assay of the supernatant.'''
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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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: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>
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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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== '''Result''' ==
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: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>
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===Chitinase A1===
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[[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>
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Standard Measurement for ChiA1.
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:'''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&plusmn;0.1, SOC was1.227&plusmn;0.007, and Plusgrow &#8545; was 0.17&plusmn;0.02.<br><br>
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[[File:Kyoto-ChiA1Standard0925.png|thumb|center|500px|Fig.1: Absorbance550 vs. glucose concentration. ''r''<sup>''2''</sup>=0.98936.]]
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:The data of Plusgrow &#8545; supports the fact that little reducing sugar is contained in this medium.
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aaa
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[[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.]]
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== '''Discussion''' ==
+
=='''4. Discussion'''==
 +
==='''Plusgrow &#8545; should be suit for the DNS assay in the first three hours.'''===
 +
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>
-
== '''Reference''' ==
+
=='''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]]
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[1] Molecular Characterization of a Gene Encoding Extracellular Serine Protease Isolated from a Subtilisin Inhibitor-Deficient Mutant of Streptmyces albogriseolus S-3253
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Moreover, We succesed modeling of chitinase activity.
 +
<html><a href="/Team:Kyoto/Digestion/Modeling">Modeling</a></html><br>
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[2]Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis
+
=='''Reference'''==
 +
[1] “Actinobacteria.” Internet: http://en.wikipedia.org/wiki/Actinobacteria [Nov. 5, 2011]
-
[3]Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites
+
[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
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[4]還元糖の定量法 (生物化学実験法)福井 作蔵
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[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
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[5]Quantitative Analysis of Cellulose-Reducing Ends
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[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
-
</div>
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[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

Latest revision as of 03:59, 6 October 2011

Contents

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.
Digestion Constrctin1.png

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.
Kyoto-digestion-DNSassay1.jpg

3. Result

This figure shows the overall of DNS assay キチナーゼ.PNG 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

  1. to plot the relationship of reducing sugar concentration and OD 550
  2. to evaluate the impact of components of media on OD 550
  3. to evaluate the impact of remeineded E.coli in media on OD 550
  4. 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.
Digestion Constraction2.png

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