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

Background

There are variety of problems all over the world today.For example, there are global warming, destruction of the ozone layer, mass extinction, energy problem, desertification and lack of water.These problems are caused by deforestation.How do we solve these problems. We can solve these environmental problems if we make paper from tree. That's why we make paper from microbe by synthetic biological way.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.

Secretion of fibrils from Agro

For this year’s project our team aimed at making the paper by using Agro bacterium. So, we confirmed whether carbohydrate developed from Agrobacterium 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

Experiment1

Sample 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.

Experiment2

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 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.

‎ ‎

　　　　　　　　　　　　　　　　Sample3　　　　　　　　　　　　　　　　　　　　　　Sample4

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

We centrifuged all the samples for 5 minutes at 4 ℃,13000 rpm.

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)

About curdlan

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. It polymerizes two position;C₁and C₃on D-glucose. Its binding position is not straight. So it is cycloid structure.

File:Biwako-nagahama curdlan's-picture yoshiharu-otaki1.png

Curdlan's property

It is insolubility in water. But it can become dispersing solution in cold water. And after high speed shuffle,the solution go to uniform dispersing solution. Moreover it become complete dispersing solution if it is mixxed by alkaline solution(avobe pH12).

CrdS

セルC

Binary Vector

fig.2
fig3.
fig4.

E.coli-ink

Another one of our project is E.coli ink.

We are planning to use Escherichia coli expressing the sperm whale myoglobin as ink. We are also trying to change the color of transgenic Escherichia coli by changing the growth and medium conditions.

Why do we use myoglobin as ink ?

Recently, fluorescent pigment is very famous and popular. It is nice to look at.

But, Many fluorescent pigment is only colouring when it’s exposed to ultraviolet rays.

We want to make pigment colouring under natural light. Thus,We decided to use myoglobin as ink.

Finally, We will draw my advisor’ face on Agrepaper with this E.coli ink !

The nucleotide sequence of myoglobin of sperm whale was already published, and We have synthesis company to synthesis the DNA fragment encoding the myoglobin.

Fig1 shows　this DNA fragment sequence. Fig1 is published on paper.

Fig1.DNA fragment encoding myoglobin.

DNA synthesis company send us the DNA fragment with pTAKN-2. pTAKN-2 is plasmid that .Thus,We get DNA fragment with pTAKN-2.

Next,We insert to pUC18 to confirm the function of this DNA fragment.

We did double digestion pUC18 and pTAKN-2 with BamHⅠand SmaⅠ.This experiment protocol shows below.