Contents 1 AgRePaper&E.coli-ink 1.1 Agrepaper 1.1.1 PROBLEM PRESENTATION 1.1.2 中島の実験 1.1.3 Curdlan's introduction 1.1.4 カードラン 1.1.5 セルC 1.1.6 Binary Vector 1.2 E.coli-ink

AgRePaper&E.coli-ink

Cellulose is used as raw material for paper, so our team experimented various ways to increase the amount of cellulose produced by agrobacterium and using it to make papers. For this we developed the different parts to insert into the system of agrobacterium. Among them are the genes used for expression of the curdlan. Similarly, genetic parts in order to increase the expression of the cellulose, along with the agrobacterium type binary vector were also developed . We are also working on recycling the produced paper by degrading the cellulose to D-Glucose using various enzymes. We worked for the preparation of the biological ink using the sperm whale's cells by genetically modification to increase amount of myoglobin. Then, we observed the change on the color of the product by altering the formation of myoglobin and the production amount of myoglobin with the insertion of T7 promoter to the cell system.

Agrepaper

PROBLEM PRESENTATION

①There are variety of issues that advanced in from today. For example Grobal warming,ozone layer destruction,biological extinction,an energy problem,desertification ,a crisis of water resources.There issues are coused by deforestation to make papers.

②How to solve it.

③Environment issues can solve if not to use wood as well as burdon the environment. We need to know “What is the paper?” to think the method.

④The main component of paper is cellulose and hemicellulose.

⑤We were confirmed by Nakasima’s experiments that Agrobacterium make cellulose. Agrobacterium can’t make hemicellulose but cardlan Agrobacterium made has potential instead of hemicellulose, Ootaki perform confimatory experiment .As a result cardlan is able to instead of hemicellulose.

中島の実験

For this year’s project our team aimed at making the paper by using Agro bacterium. So, we confirmed whether carbohydrate developed from Agro bacterium could be used as the raw material for making paper.

Materials

Agro bacterium tumefaciens C58　

Agro bacterium tumefaciens C58　bacterial liquid

　　　　＋LB medium　　　　 　 2 ml

　　　　＋ampicillin　　　　　　 　2 µl

　　　　＋Agro bacterium tumefaciens C58

Congo red

LB medium

　　　　＋1.0％(w/v)　 Triptone　　　　 2 g

　　　　＋0.5％(w/v)　 yeast extract　 1 g

　　　　＋1.0％(w/v)　　NaCl　　　　　 2 g

Sample 1 curdlan 15 µl　　　　 ＋Congo red 5 µl　＋Agro bacterium precipitationSample 1 curdlan 15 µl　　　　 ＋Congo red 5 µl　＋Agro bacterium precipitationSample 1 curdlan 15 µl　　　　 ＋Congo red 5 µl　＋Agro bacterium precipitation

Sample 2 de-ionized water 15 µl　＋Congo red 5 µl　＋Agro bacterium precipitation

We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).

As a result, Sample 1 had red precipitates. Sample 2 rationally melted.

So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.

Next, we found that carbohydrate could be produced when soy milk is added to the fluid bacteria

So, we experimented for the confirmation of the above findings.

Sample 3　LB medium 7 ml ＋Agro bacterium＋soy 40 µl

Sample 4　LB medium 7 ml ＋Agro bacterium

Sample 5　LB medium 7 ml　　 　 ＋soy 40 µl

Sample 6　LB medium 7 ml

We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).

As a result, Sample 1 had red precipitates. Sample 2 rationally melted.

So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.

Sample 2 de-ionized water 15 µl　＋Congo red 5 µl　＋Agro bacterium precipitation

We centrifuged Sample 1 and 2 in 5 minutes with cooled centrifuges(4 ℃,13000 rpm).

As a result, Sample 1 had red precipitates. Sample 2 rationally melted.

So, we confirmed the presence of carbohydrate in the precipitates obtained from Agro bacterium.

Next, we found that carbohydrate could be produced when soy milk is added to the fluid bacteria<p/> <p>So, we experimented for the confirmation of the above findings.

Sample 3　LB medium 7 ml ＋Agro bacterium＋soy 40 µl

Sample 4　LB medium 7 ml ＋Agro bacterium

Sample 5　LB medium 7 ml　　 　 ＋soy 40 µl

Sample 6　LB medium 7 ml

We cultivated Sample 3 ~ 6 at 28 ℃.for 12 hours

Then, Sample1 and 2 produced a white film like substance but Sample 3 and 4 did not.

We put the white films from Sample 1 and 2 into micro-tubes.

Sample 7　 supernatant(Sample 1) 15 µl　＋Congo red 5 µl

Sample 8　 supernatant(Sample 1) 15 µl　＋Congo red 5 µl　＋de-ionized water 1 ml

Sample 9　 supernatant(Sample 2) 15 µl　＋Congo red 5 µl　

Sample 10　supernatant(Sample 2) 15 µl　＋Congo red 5 µl　＋de-ionized water 1 ml

Sample 11　Congo red 5 µl　＋de-ionized water 1 ml

As a result, Sample 7 ~ 10 gave red precipitates but Sample 11 did not.

So, we confirmed Sample 7 ~ 10 had carbohydrate.

Through these experiments, we confirmed that Agro bacterium can discharge carbohydrate without soy milk.

Reference

1, Laboratory maintenance of Agrobacterium.

(http://europepmc.org/articles/PMC3350319)

2, Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens (Jinwoo Kim.¤, Jason E. Heindl., Clay Fuqua)

3, Kirin Kyowa Foods Company (http://www.kirinkyowa-foods.co.jp/products/curdlan/)

4, Howard Hughes Molecular Biology Summer Research Program Poster, Austin, TX , August, 1995 Microcopy of Curdlan Structure

5, Iain M. Cheeseman and R.Malcom Brown,Jr, Department of Botanty , The University of Texas at Austin,TX,78713

(http://www.botanty.utexas.edu/facstaff/facpages/mbroun/ongres/icheeze.htm)

Curdlan's introduction

Agrobacterim makes carbohydrate chain. For exampe it makes curdlan on culture midium.We'll explain curdlan on this page.

<About curdlan> It is fermentative polysaccharide that microorganizm makes.

カードラン

セルC

Agro Notebook

5/31 Cloning of CelC and Restriction Enzyme

By Koki Tsutsumi

CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI.

Binary Vector

E.coli-ink