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Team:Kyoto/Digestion
From 2011.igem.org
Contents |
Project Digestion
Introduction
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.
Method
Construction
Assay
Serine Protease
- Experiment 1 : Skim milk-hydroryzing assay
- 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.
- Experiment 2 : Measurement of enzyme activity
Chitinase A
- Experiment 1
- Experiment 2 : Dinitrosalicylic acid assay
- This assay is based on this fact: 3,5-dinitorosalicylic acid 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.
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 into 3,5-dinitorosalicylic acid solution (how to prepare? :プロトコルへのリンクを貼る) and assay the absorbance in 550 nm. 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
