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Project Digestion

Introduction

An insect body is covered with a hard integument containing mainly chitin. To decompose the integument, we used ChiA gene, which encode chitinase. In order to measure the chitinase activity of the culture supernatant, we evaluated the effects of the medium and cell growth.

Streptomyces is a kind of prokaryotic bacteria which decompose bodies in the nature. We extract chitinase gene from this bacterium and introduce into Escherichia coli. The 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, and seek equations of production of chitinase and N-acetylglucosamin.

Method

Construction

We created following construction to allow secretion of chitinase, chiA1. This gene is regulated by strong 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.
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Assay

We performed 3,5-Dinitrosalicylic acid assay (DNS assay), because this assay takes a little time, costs a little money and was used in the previous article measuring chitinase activities [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.

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 in direct proportion to the amount of 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.

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

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
   

1. Standard Measurement for ChiA1. In order to measure the product of the enzyme reaction, N-acetylglucosamin, quantitatively, we prepared the standard curve using glucose as standard. It is known that the correlation between glucose concentration and the absorbance can be plotted in linaer.

From the result, a strong correlation between glucose concentration and its A₅₅₀ was observed.

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

2. Consideration of medium and growth of E.coli.

To get accurate values of absorbance, it is needed to consider the remained cells in supernatant. Since we incubate the supernatant for hours for the enzyme reaction, cells can grow, produce chitinase and consume glucose, N-acetylglucosamin, etc; this can influence to DNS assay.

2-1 The absorbance of supernatant itself.

In this experiment, we checked if cells were still observed in the supernatant and assayed the growth behavior of cells. Figure 2 shows the A₅₅₀ of supernatant with time. The absorbance values of M9 were increased slowly and they were smaller than 0.2 during the incubation. The absorbance values of SOC were also increased slowly and they were higher than those of M9. The absorbance of values of Plusgrow Ⅱ were increased obviously between the 3h and 5h.

Fig.2:

2-2 The DNS assay of supernatant.

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

In this experiment, we evaluated the influence of cells and its growth to the DNS assay. In other words, we confirmed if cells consume glucose and affect the 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.

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.

From figure 2 and 3, the cell growth didn't affect to the absorbance of supernatant. 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 and this is the reason that

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.

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.

Discussion

Modeling

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.

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.

To overcome these barriers, we decided detail plan of our assay. 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, the increase of absorbance was lowest and there were fewest E.coli after 5 hour culture. So we choose M9 as the medium we will used in our assay. Another barrier, interruption of remained E.coli, can be avoided by the following way, conducting DNS assay before chitin is applied and after chitin is decomposed. The difference of these two assays’ data will show the real activity of chitinase.

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)” 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