Difference between revisions of "Team:Tokyo Tech/Description"

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<h2> Project Description</h2>
 
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<p>Our Team Tokyo_Tech is currently working on the following four projects:</p>
 
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<p>Ⅰ. Representing fashion show and Othello with the interaction among <i>E.Coli</i>.
 
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<p>Ⅱ. Engineering the <i>E.Coli</i> which can secrete and absorb protein.
 
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<p>Ⅲ. Engineering the <i>E.Coli</i> which can increase the efficiency to allocate phosphorus fertilizer and synthesize a variety of plant hormones.
 
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<p>Ⅳ. Representing the famous fairy tale, Snow White with the interaction among <i>E.Coli</i>.
 
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<p>Ⅰ. We have developed the mechanism about coating the surface of <i>E.Coli</i> , and run the fashion show of <i>E.Coli</i> with various kinds of fluorescence. It is expected that the development of proteins which bind with specific antibody or cell targeted would be applied to determining the location of the target, establishing the system to control the motion of the target and also microbe sensor for the specific substance. This system will be applied in various fields such as Biology and Medicine. In our opinion, the control of multiple motions among <i>E.Coli</i> will be used as a device of microbe sensor by combinating <i>E.Coli</i> and hardware.
 
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<p>Ⅱ. We aim to develop the system of <i>E.Coli</i> which can secrete a large amount of target protein.  because in order to synthesize and collect target protein, we have to break down the whole <i>E.Coli</i>, It‘s a time-consuming process to get the purified target protein. However, if the system secreting the target protein efficiently can be established, we’ll be able to produce the target protein without killing <i>E.Coli</i> and reduce the cost of production.
 
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<p>Ⅲ. We are trying to produce phosphoric acid from phosphate in E. Coli which is hardly soluble and not absorbed by plants. Through this process，it will be able to increase the efficiency of phosphorus fertilizer’s allocation and to alleviate the depletion of phosphate rock. In addition, we aim to grow vegetables in a short term by synthesizing plant hormone by <i>E.Coli</i>.
 
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<p>Ⅳ.  We plan to apply AHL-degrading enzyme and toxin used for representing Snow White by <i>E.Coli</i> in the practice. AHL-degrading enzyme can be applied to preventing the generation of biofilm. In our opinion, the death of malignant cells can be induced by inducing the expression of toxin gene. According to the mathematical modeling about competition and balance of bacteria, we are still studying about the analysis of the competition among bacteria populations <i>in vivo</i>, river and soil.
 
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           <h2 id="Summary" class="smalltitle">0. Summary</h2>
 
           <h2 id="Summary" class="smalltitle">0. Summary</h2>
 
           <p class="text">When we tried to represent Snow White with TA system and Quorum Sensing, we faced two problems. First, we could access too little data of TA system to work on our project. Second, simulation of our final genetic circuits revealed that Rhl system was too weak to function as well as Lux system and our final circuits could not work well. So we decided to characterize the existing TA system and rhlR parts, and improve the past improved Prhl in order to represent Snow White as faithfully as we could. As a results, the data we obtained makes easy to use these existing TA system and rhlR parts. Furthermore, our original improved Prhl expands the possibility to use Quorum Sensing.</p><br>
 
           <p class="text">When we tried to represent Snow White with TA system and Quorum Sensing, we faced two problems. First, we could access too little data of TA system to work on our project. Second, simulation of our final genetic circuits revealed that Rhl system was too weak to function as well as Lux system and our final circuits could not work well. So we decided to characterize the existing TA system and rhlR parts, and improve the past improved Prhl in order to represent Snow White as faithfully as we could. As a results, the data we obtained makes easy to use these existing TA system and rhlR parts. Furthermore, our original improved Prhl expands the possibility to use Quorum Sensing.</p><br>
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           <h2 id="TA system" class="smalltitle">1. TA system</h2>
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           <h2 id="Characterization of TA system" class="smalltitle">1. Characterization of TA system</h2>
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              <a name="Characterization of TA system"></a>
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              <h3 id="21" class="sub5">2.1. "Background”for our Characterization of TA system</h3>
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              <p class="text2">TA system is the function that regulates cell growth on the genomic DNA of many microorganisms.
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  So far, iGEM 2014 team ULB-Brussels, iGEM 2013 team UGent had studied about ccdB-ccd, iGEM 2014 team Wageningen UR about Kid-Kis and Zeta-Epsilon, iGEM 2015 team UMaryland about Hok-Soc. Almost all the iGEM teams dealing with TA system aimed to use it as Kill Switch or substitution for antibiotics. On the other hand, we aim to study the interaction of toxin and antitoxin in a cell. Besides some teams failed to show enough data of TA system assay. Although UCSF 2012 experienced the communication through TA system including mazEF system, they did not introduce toxin and antitoxin into a cell and could not show mazEF system assay data. Our results show that the cell growth can be controlled by mazEF system.</p>
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              <p class="text2"> iGEM 2016 team Tokyo_Tech has studied about mazEF system (MazF : toxin , MazE : antitoxin) that has been researched well as TA system.
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</p><br>         
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              <h3 id="21" class="sub5">2.2. Summary of the experiment</h3>
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              <p class="text2"></p><br>
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              <h3 id="21" class="sub5">2.3. Results</h3>
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              <p class="text2">The level of GFP expression of (a)~(d) are shown below.</p><br>
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              <h3 id="21" class="sub5">2.4. Discussion</a></h3>
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              <p class="text2"> The expression level of the gene under Prhl depends on not only Prhl activity but also the type of the rhlR. In other words, this characterize shows that the combinations of Prhl and rhlR can control the expression level of the gene under Prhl. It gives you more choices which Prhl and rhlR you use.</p>
 
                  
 
                  
 
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           <h2 id="Characterization of rhlR" class="small title">3. Characterization of rhlR</h2>
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           <h2 id="Characterization of rhlR" class="smalltitle">3. Characterization of rhlR</h2>
 
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               <h3 id="21" class="sub5">2.1. "Background”for our Characterization of rhlR</h3>
 
               <h3 id="21" class="sub5">2.1. "Background”for our Characterization of rhlR</h3>

Revision as of 02:01, 15 October 2016

Team:Tokyo_Tech - 2016.igem.org
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Team:Tokyo_Tech

Tokyo_Tech

Description

　　

Contents

0. Summary

1. Characterization of TA system

2. Improvement of Prhl

3. Characterization of rhlR


　
　　

0. Summary

When we tried to represent Snow White with TA system and Quorum Sensing, we faced two problems. First, we could access too little data of TA system to work on our project. Second, simulation of our final genetic circuits revealed that Rhl system was too weak to function as well as Lux system and our final circuits could not work well. So we decided to characterize the existing TA system and rhlR parts, and improve the past improved Prhl in order to represent Snow White as faithfully as we could. As a results, the data we obtained makes easy to use these existing TA system and rhlR parts. Furthermore, our original improved Prhl expands the possibility to use Quorum Sensing.


1. Characterization of TA system

2.1. "Background”for our Characterization of TA system

TA system is the function that regulates cell growth on the genomic DNA of many microorganisms. So far, iGEM 2014 team ULB-Brussels, iGEM 2013 team UGent had studied about ccdB-ccd, iGEM 2014 team Wageningen UR about Kid-Kis and Zeta-Epsilon, iGEM 2015 team UMaryland about Hok-Soc. Almost all the iGEM teams dealing with TA system aimed to use it as Kill Switch or substitution for antibiotics. On the other hand, we aim to study the interaction of toxin and antitoxin in a cell. Besides some teams failed to show enough data of TA system assay. Although UCSF 2012 experienced the communication through TA system including mazEF system, they did not introduce toxin and antitoxin into a cell and could not show mazEF system assay data. Our results show that the cell growth can be controlled by mazEF system.

iGEM 2016 team Tokyo_Tech has studied about mazEF system (MazF : toxin , MazE : antitoxin) that has been researched well as TA system.


2.2. Summary of the experiment


2.3. Results

The level of GFP expression of (a)~(d) are shown below.


2.4. Discussion

The expression level of the gene under Prhl depends on not only Prhl activity but also the type of the rhlR. In other words, this characterize shows that the combinations of Prhl and rhlR can control the expression level of the gene under Prhl. It gives you more choices which Prhl and rhlR you use.


2. Improvement of Prhl

2.1. "Background”for our improvement of Prhl

We simulated our final genetic circuits and found that the circuits did not work, because Prhl activity was too weak compared to Plux. (see the Model page and the AHL Only Assay page). We therefore considered using the improved Prhl (BBa_K1529310, BBa_K1529300) established by iGEM 2014 Tokyo_Tech, but we noticed that they were inappropriate for two reasons: crosstalk to C12 and type of rhlR (written below). Then, we decided to improve wild type Prhl.


2.2. Summary of the experiment

By introducing a single point mutation into wild type Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and obtained the Prhl mutants of which promoter activity was stronger than wild type Prhl, and named it Prhl(Noticeable Mutant) (BBa_K1949060), hereafter referred to as Prhl(NM), which suited our goal.


2.3. Results

The SN ratio of Prhl(NM) was higher than that of Prhl(LR) and Prhl(NM) did not crosstalk to C12 unlike Prhl(LR).


2.4. Discussion

Many iGEM teams used AHL and probably this tendency continues. When you use AHL, you will find that promoter strength is quite different depending on the type of AHL and it bothers you to design genetic circuits, for example, including Plux and Prhl. Prhl(NM) has almost as same strong activity as Plux. Furthermore, Prhl(NM) has higher specificity to C4 than Prhl(LR). Prhl(NM) can be said that it expands the possibility of using various combination promoters depending on AHL.


3. Characterization of rhlR

2.1. "Background”for our Characterization of rhlR

When we used two type of rhlR, one was LVA-tagged and the other was normal, we found that gfp introduced with rhlR barely expressed but gfp introduced with rhlR-LVA expressed greatly. We decided to find this cause.


2.2. Summary of the experiment

Measure RFU of GFP of the E. coli containing (a) Pcon-rbs-rhlR-LVA (pSB6A1), Prhl(LR)-rbs-gfp (pSB3K3), (b) Pcon-rbs-rhlR (pSB6A1), Prhl(LR)-rbs-gfp (pSB3K3), (c) Pcon-rbs-rhlR-LVA (pSB6A1), Prhl(RL)-rbs-gfp (pSB3K3) or (d)Pcon-rbs-rhlR(pSB6A1), Prhl(RL)-rbs-gfp (pSB3K3). And we found that gfp expresses much if it introduced with rhlR-LVA even though using stronger Prhl than wild type Prhl. Thls is because that RhlR, which represses Prhl without C4, tagged LVA is prone to be degraded and the concentration of RhlR in a cell decreases then the repression becomes weak.


2.3. Results

The level of GFP expression of (a)~(d) are shown below.


2.4. Discussion

The expression level of the gene under Prhl depends on not only Prhl activity but also the type of the rhlR. In other words, this characterize shows that the combinations of Prhl and rhlR can control the expression level of the gene under Prhl. It gives you more choices which Prhl and rhlR you use.

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