Team:KIT-Kyoto/Project/Abstract

Abstract

"E.coli Pen": Draw with your own color.

Our team, KIT-Kyoto suggests an “E.coli Pen” as a new Art Tool. This brand-new pen uses no ink but medium in which genetically modified E.coli has been cultured. The Pen is able to express more than four colors in various intensities with single bacterial culture. This will be achieved by constructing plasmids carrying genes coding for four different fluorescent proteins under the control of seven promoters having different sensitivity to oxidative stress. The E. coli carrying these plasmids will produce different colors with various intensities by differentially responding to the gradient of hydrogen peroxide treatment. Different from previous passive BioArt in iGEM, the genetically engineered “E. coli Pen” provides an active and wonderful tool for us to purely enjoy the Art having a feeling for biotechnology.

Introduction

A brand new form of art has emerged with the revolution of biology in the last ten years, this is Bio-Art. Adding new artistic elements, such as cells, DNA, genomes, proteins, enzymes, etc., this new form of artistic expression has as medium living matter, and the studios of the artists are biological laboratories, while high end technologies of genetic engineering, tissue culture and many others are their new painting brushes.

BioArt is considered by most artists to be strictly limited to “living forms,” although there is some debate as to the stages at which matter can be considered to be alive or living. Creating living beings and practicing in the life sciences brings about ethical, social and aesthetic inquiry.

Here, we aim at a completely new style of BioArt. In hitherto BioArt, the appreciator enjoys a work in the conventional way, that is he/she appreciates an artist’s resulting piece looking or touching it. The new style of BioArt we propose here is the enjoyment of making art itself. With the “E. Coli Pen” that we propose here the appreciator himself becomes part of the artistic work he is drawing enjoying the fascination of making the work and the work itself.

Our team, KIT-Kyoto has designed and carried out the project taking account of the key word "Make the science and technology more accessible to the public".

    Recently, it is getting more and more serious problem that people think the science is too difficult to understand. Moreover, it is expected that people are moving away from the science as the science becomes more complicated. Therefore, to overcome such a problem, we would like to offer more opportunities for the public to interact with the science especially for the person who is not interested in the modern science. 
    In deciding our project, we thought it is the most important that anyone can readily enjoy the science and technology. We therefore decided to focus on "Bio art" in order to invent the artistic products by using biotechnology, since Art is more accessible to the public. Moreover, unifying the two apparently distant academic fields "Science" and "Art" is an educational philosophy of our University, Kyoto Institute of technology. 

(More learn BioArt とかでリンクを飛ばす)

    However, an already existing bio art does not meet the key word “Make the science and technology more accessible”, because that the preexisting bio art needs special equipment and the deep knowledge of biotechnology etc. We therefore decided to work on the development of a new art tool by which people could enjoy the bio art easily. 
    Here we can propose a novel art tool, “E.coli pen” which draws paintings with innumerable colors as inks of the pen that are produced by a single E. coli bacteria. "E.coli pen" would further develop the field of the bio art as a new art tool. Furthermore it is useful as an educational toy, since people can enjoy drawings with the heavenly color with happiness and at the same time they can study the molecular mechanism how the colors are made by genetic engineering.

   We will summarize how to make "E.coli pen" in the following section. 

1. How to make an innumerable color in E. coli bacteria 1-1. Use of light's three primary colors Principle of light’s three primary colors (red, green, and blue) can be applied to create an innumerable color. The fluorescent protein can be used as a color source. Red Fluorescent Protein (RFP) can be used for Red, GFP for Green, and CFP for Blue. The fluorescent color is originated when ultraviolet rays is irradiated. These proteins are widely used in the field of the genetic engineering as the reporter gene. Therefore once we obtain the red, green, and blue fluorescent proteins in our hands, we can create an innumerable color by simply mixing these three fluorescent proteins with various ratios. At first we tried to produce the red, green, and blue fluorescent proteins in E. coli.

    To achieve this, the multi-gene cassette shown in the Figure 1 was newly designed. 

-P-RBS-GFP-TM-TM-P-RBS-RFP-TM-TM-P-RBS-CFP-TM-TM-

    In this multi-gene cassette, the genes encoding GFP, RFP and CFP are placed in a tandem array. Each gene can be controlled by different promoter that has different activity. In order to create different color, fluorescent proteins have to be produced in different ratios. This can be achieved by utilizing promoters having different activities. 

1-2 Selection of appropriate promoter Then, what kind of promoter is actually appropriate for this purpose? The promoter activity has to be controlled by some regulating substances. The easily available substance that had not been used before in iGEM would be suitable for the purpose., If the substance is related to some human diseases or aging, the promoter responding to the substance may be used for diagnosis of the disease or aging, since the fluorescent proteins under the control of the promoter can be used as markers. From these standpoints we decided to use promoters responding to hydrogen peroxide. Hydrogen peroxide is one of the reactive oxygen species that oxidize nucleic acid, lipid and protein to result in aging of human cells and various diseases. Although effective use of oxygen is acquired during evolution, the defense system to the active oxygen generated by consumption of oxygen also has been acquired in the aerobe. There are a set of genes that are involved in this defense system. These genes can be induced by hydrogen peroxide. We have utilized the promoters of these genes to induce the fluorescent proteins.

1-3 Mechanism of hydrogen peroxide response E. coli has two defense mechanisms against the active oxygen . One is OxyR response, and another is SoxRS response.

    In the OxyR response, OxyR works as a transcription factor. OxyR regulates activation or repression of the gene expression. Without active oxygen, OxyR is in an inactivated form. And with active oxygen, OxyR get activated and the activated OxyR can bind to specific DNA sequence. OxyR binding site are found in promoters of a set of genes that respond to the active oxygen. In these active oxygen response genes, we focused on the promoter of ahpC dps, oxyR, sufA, and yaiA. These genes carry no recognition sequences of EcoRⅠ, XbaⅠ, SpeⅠ,and PstⅠ restrict enzymes. These restriction enzyme sites would be used for cloning into the psB1C3. The promoters of these genes cloned into psB6A1 are listed in Biobrick site.
    In the SoxRS response, SoxR works as a transcription factor. SoxR is constantly expressed in cells. SoxR binds to the promoter of SoxS, and represses its transcription without hydrogen peroxide. With hydrogen peroxide, SoxR activates the SoxS gene transcription. SoxS also works as a transcription factor of two or more downstream genes responsible to the hydorogen peroxide (具体的な遺伝子を挙げる). In these hydrogen peroxide response genes, we focused on the promoter of acrAB and soda. Both of these genes have no sequence of EcoRⅠ, XbaⅠ, SpeⅠand PstⅠ restrict enzyme sites. These restriction enzyme sites would be used for cloning into the psB1C3. The promoters of these two genes cloned into psB6A1 are listed in Biobrick site.

1-4 Active oxygen response promoters Out of these seven active oxygen response promoters, we here shows the structure of ahpC and sufA promoters.

・ahpC promoter (臼井君から)図とその説明を入れる。 ahpC-F are the genes involved in oxidative stress defense. Transcription of these genes is regulated in response to H2O2. The ahpC and ahpF genes encode the small subunit and large subunit of alkyl hydroperoxide reductase, respectively. The complex of AhpC and AhpF reduces alkyl hydroperoxide. The ahpC-promoter is controlled by the OxyR protein, an oxidative stress responsive transcription factor. OxyR is constitutively expressed but activated only under oxidative stressed conditions caused by H2O2. The activated OxyR binds to the promoter region of the ahpC gene and promotes its transcription.

・sufA promoter(臼井くんから) sufA promoter is controlled by some transcription factors including OxyR, regulates the transcription of sufABCDSE operon encoding an alternative Fe-S cluster assembly system in E.coli. We also constructed expression systems in which the sufA promoter controls the transcription of fluorescent protein genes and measured the fluorescent intensity.

We thought an innumerable color for us to able to produce by examining these promoter and the survival rate of E. coli bacteria by the density of the active oxygen E. coli bacteria.

2. How is the pen that efficiently mixes the active oxygen and E. coli made? The E.coli pen changes the mixing rate for E. coli culture medium and hydrogen peroxide. The volume of each solution, placed in different tubes within the pen, can be regulated by the frequency and intensity of pressing the pen's piston which can be interchanged among both tubes. For example, to make red ink, one presses the pyston in the E. coli tube three times and then interchanges to the hydrogen peroxide tube and presses the piston twice. Or, to use green ink, press the E. coli tube five times and then the hydrogen peroxide tube only once. In this way, one is able to produce any color. This is a spectacular way of producing new colors that any conventional pen can not realize. The movie of the E.coli pen is shown in the first page of wiki of our team.

Results & Discussion

Materials & Methods

References