Team:Tokyo-Nokogen

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<title>Tokyo-NokoGen</title>
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<p align="center"><img src="../NokoGen_logo.jpg" width="40%" height="40%" alt="" align="middle"></p>

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<b><u>Introduction of our team</u></b><br>

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<p style="text-indent: 1em;">We belong to the Department of Biotechnology at Tokyo University of Agriculture and Technology (TUAT) in Japan. Our team comprises ten undergraduate students, four instructors and an advisor. This is the first time for us to take participate in iGEM. Prof. Sode informed us about iGEM competition, and we girded up our loins to build 'Tokyo-NokoGen'. All the members have a great deal of interest in synthetic biology although we don't major in synthetic biology.Trying to achieve the goal of our project, we would like to realize how wonderful synthetic biology is. It is attractive for us because synthetic biology is capable of unrestricted design and construction of a novel biological system by bottom-up approach starting from almost nothing, different from "genetic engineering" that is our major. We think it is essential to unify biotechnology with other research fields such as system engineering, mathematics and bioinformatics because the biological system is so complicated. This correlation of different fields of study is what we aim to do in our department of Biotechnology in TUAT.We are glad to have a precious experience to learn how to develop our project. In this year, we will corporate with NYMU-Taipei at National Yang Ming University in Taiwan. We have already had a first live meeting and gotten to know each other. From now on, we would like to compete with each other and construct attractive genetically engineered machine.With renewed dreams and hope, we have started our projects !
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<p align="center"><b><u>Members</u></b><br>

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<b><i>Instructors</i></b><br>
Stefano Ferri<br>
Kazunori IKEBUKURO<br>
Wakako Tsugawa<br>
Jihoon Kim<br>

<b><i>Students Member</i></b><br>
Yoshiyuki MURAKAMI<br>
Yuki Anze<br>
Nasa SAVORY<br>
Daichi Nagae<br>
Hideyo Umeta<br>
Daisuke HIRAOKA<br>
Yasuhiko SASAKI<br>
Kenji Kakubori<br>
yusuke miyamoto<br>
Nao IWATA<br>

<b><i>Advisors</i></b><br>
Koji Sode<br></p>

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<b><u>Overview</u></b><br>

<p style="text-indent: 1em;">In biochemistry experiments, expression and getting objective protein is popular. But the procedure is almost vexatious complication due to need many solutions and centrifugations. So we hope to solve this trouble to construct alternative easily system with synthetic biology. The system we named Escherichia coli auto protein synthesizer. It has three components.
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1.	<u>Two colors light switches</u><br>
2.	<u>Aggregation</u><br>
3.	<u>Signal count system</u><br>
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A figure that shows procedures this system is below. We only have to irradiate E.coli three times and purification to get proteins in it. 

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<p align="center"><img src="https://static.igem.org/mediawiki/2009/6/6b/ESCAPES_system.png" width="70%" height="70%" alt="" align="center"></p>

<p align="center"><img src="https://static.igem.org/mediawiki/2009/c/cf/ESCAPES_cascade.png" width="80%" height="80%" alt="" align="middle"></p>

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<p style="text-indent: 1em;">By first induction with red light, objective protein is expressed in E.coli. Then same induction repeated, but Antigen 43 that is protein for aggregation E.coli is expressed by signal count system. Once medium replaced, we think that we use blue light with phycoerythrine as receptor. Thereby, holin and endolysin are expressed and lysis Ecoli. Finally, by purification of objective protein, this system is completed.
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<b><u>Blue Light Receptor - Blue/red light switch</u></b><br>

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<p style="text-indent: 1em;">Our team,Tokyo-NokoGen, focuses on the complementary chromatic adaptation (CCA), which occurs in many species of cyanobacteria. During CCA, cyanobacteria change their phenotypes, switching between red and blue-green color in response to shifts in the ratio of red light to green light in their environment. Based on this CCA, we are trying to engineer photo-switching E. coli that identifies red and blue light and regulates expressions of genes in response for each light signal.
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<p style="text-indent: 1em;">In cyanobacteria, CCA results in alterations of the protein composition of the light-harvesting phycobilisomes to maximize light absorption. In red light, cyanobacteria express red light receptor protein phycocyanine, and cyanobacteria express phycoerythrin as blue light receptor in response to blue light. These photoreceptors respond to each color and alter expression of specific genes.
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<p style="text-indent: 1em;">In a previous iGEM competition, Levskyaya et al. designed a bacterial system that is switched between different states by red light. In this system, phycocyanobilin biosynthetic genes (BBa_I15008 and BBa_I15009) for chromophore formation and red light responsive domain fused EnvZ (BBa_I15010) that phosphorylates endogenous OmpR as the second signal have been used as red light sensing parts (Fig. 1).
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/1/16/Light_1.png" width="387" height="278" alt="" align="center"></p>

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<p style="text-indent: 1em;">Now, in our project, we plan to add blue light sensing parts that function in E. coli into the Standard Registry. We are focusing on phycoerythrin from cyanobacteria as a blue light receptor and trying to clone and express phycoerythrobilin synthesis enzyme gene (pebA), apo-PE gene (cpeB), and phicoerythrin linker gene (cpeC) in E. coli (Fig. 2). To apply these blue light sensing parts together with the red light sensor, we are also considering adding new two-component signaling system into the Standard Registry.
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/e/e3/Light_2.png" width="385" height="316" alt="" align="center"></p>

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<p style="text-indent: 1em;">We are expecting to contribute to iGEM and synthetic biology community with our success in this project. With our success, we hope to improve the application potential of previous red sensing parts as a blue/red light switch (Fig. 3).
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/0/0d/Light_3.png" width="389" height="269" alt="" align="center"></p>

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<b><u>RTC counter - signal counting switch</u></b><br>

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<p style="text-indent: 1em;">Our group's objective is designing a signal counting switch to simply processes in vie of automation. This would allow us to use only one signal (e.g., red light) in our team's ESCAPE system instead of using a different signal for each step. We focus on riboregulated transcriptional cascade (RTC) counter; which is based on a transcriptional regulation.
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<p style="text-indent: 1em;">The concept is illustrated below (Fig. 1) for the two target genes A and B. A constitutive promoter drives transcription of T7 RNA polymerase (RNAP), whose protein transcribes gene A and T3 RNAP, which are regulated by a T7 promoter. Then, T3 RNAP transcribes gene B, which is regulated by a T3 promoter. However, the translation of all these genes is controlled by riboregulators, whose cis and trans elements silence and activate posttranscriptional gene expression, respectively. A cis-repressor sequence (cr), located between the transcription start site and the ribosome-binding site (RBS), is complementary to the RBS. This complementarity causes it to from a stem-loop structure upon transcription and prevent binding of the 30S ribosomal subunit to the RBS, thus inhibiting translation. A short, transactivating noncoding RNA (taRNA), driven by an inducible promoter, binds to the cis repressor in trans, thus relieving RBS repression and allowing translation. With this riboregulation, each node (i.e., gene) in the cascade requires both independent transcription and translation for protein expression (Fig. 2). This cascade is able to count frequency of the signal by expressing a different protein in response to each time the signal is applied.
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/a/ac/Counter_1.png" width="453" height="176" alt="" align="middle"></p>

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<p align="center"><img src="https://static.igem.org/mediawiki/2009/d/d3/Counter_2.png" width="609" height="176" alt="" align="middle"></p>

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<p style="text-indent: 1em;">We plan to construct a counter system using a red light-responsive promoter (or a blue light-responsive one) to detect the number of colored light exposures. The target genes that will be expressed in sequence in response to the light signal are antigen 43 (gene A) and holin and endolysin (gene B). This signal counting switch will help make our team's ESCAPE system very convenient.
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<p style="text-indent: 1em;">We are expecting to contribute to iGEM and the synthetic biology community with this project. By our success, we hope to enable complex synthetic programming for a variety of biotechnology applications.
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【Parts】

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PBAD promoter (BBa_I13453), T7 promoter (BBa_I719005), Riboregulator Lock 1[cs+RBS in Fig. 1] (BBa_J01010), Riboregulator key 1[taRNA in Fig. 1] (BBa_J01008), T7 RNA polymerase (BBa_I2032), GFP　(BBa_E0040), RFP(Part:BBa_E1010), Constitutive promoter (BBa_J23100), T3 promoter, T3 RNA polymerase

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【Reference】

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・Ari E. Friedland, et al., Science 324, 1199 (2009).
・F. J. Isaacs et al., Nat. Biotechnol. 22, 841 (2004).

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<b><u>Aggregation</u></b><br>

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<p style="text-indent: 1em;">We want to make a system to harvest E.coli easily by E.coli autoaggregation after protein abundant expression. If the E.coli autoaggregation occurs in culture media, we don't need a step of centrifugation.</p>

<p style="text-indent: 1em;">Antigen 43 (Ag43) is a key protein in this system. This protein is able to mediate autoaggregation and ?occulation of E. coli cells in static cultures. In E. coli, expression of Ag43 induces autoaggregation and settling of cells from　standing liquid cultures. This phenomenon is mediated by Ag43-Ag43 interaction.
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<p style="text-indent: 1em;">We plan to express Ag43 in E. coli by red light induction as be shown earlier, we can get fungus body easily.</p>

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<p align="center"><img src="https://static.igem.org/mediawiki/2009/1/1a/Agreation_1.png" width="131" height="178" alt="" align="middle"></p>

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<b><u>The Lysis System</u></b><br>

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<p style="text-indent: 1em;">Our team, Tokyo-NokoGen, focuses on the lysis system of the phage. We proposes a novel protein purification procedure, called ESCAPES(Escherichia Coli Auto Protein) Synthesizer. In the previous procedure, we must crush the recombinant cells by sonicate or French press to get a target protein. However, with the lysis system, we do not have to do that. We focuses on this merit and will apply this system to the process after the aggregation of E.coli and construct our project, ESCAPES(Escherichia Coli Auto Protein) Synthesizer.
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<p style="text-indent: 1em;">The lysis system is carried out by two specific proteins, Holin and Endolysin. Both of their genes(hol and lyt: Part:BBa_K124003) are encoded on pharge  1/6 DNA(Fig.1A). Holin releases Endolysin to the periplasm and Endolysin acts as peptide glycan hydrolases.
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/8/83/Lysis_1.png" width="363" height="225" alt="" align="middle"></p>

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<p style="text-indent: 1em;">Expression of these lysis genes in Eschericia coli was done to investigate the effect of these lysis products on the growth of E. coli BL21(DE3) havoring pET R and pACEThol, which contains the lyt and hol, respectively(Fig.2A1). 
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/3/33/Lysis_2.png" width="272" height="196" alt="" align="middle"></p>

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<p style="text-indent: 1em;">As a further experiments, Plate assay for induction in Streptomycetes was done. Expression was induced with different amounts of thiostrepton applied in the disk on the surface of the plate. The expression of the lyt and hol genes from plasmid pIJlythol showed the lysis of the S. aureofaciens along the induction levels(Fig.3D).
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<p align="center"><img src="https://static.igem.org/mediawiki/2009/5/5e/Lysis_3.png" width="187" height="185" alt=""></p>

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<p style="text-indent: 1em;">We are expecting that our ideas and project with this system contribute to iGEM and synthetic biology.
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【Reference】

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・Jarmila Farkas ovska´ A Andrej Goda´ny et al. Curr Microbiol(2008)

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<b><u>Schedule</u></b><br>

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May-------Build up Team 'Tokyo-Nokogen'<br>
June------Brain storming, go to work shop for Asia<br>
July------Summarize the ideas, design circuits, order genes<br>
August----Construct of plasmid, confirm function of each parts<br>
September-Construct each device as sub-project, build up the ESCAPES system<br>
October---Evaluate the systems, prepare for the presentation<br>
November--Jamboree<br>

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