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

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Revision as of 02:53, 19 October 2016

Project

1. Introduction

When you were very young, before going to sleep, have you ever asked your mother, “Mommy, read some picture books to me, please.”? You may have been read it aloud at least once.
Most stories for generations have lessons. The lessons from Snow White story are mainly the following two points.
Ⅰ It is not good to show off or to be jealous.
Ⅱ Do not trust unfamiliar people blindly.
It is not enough to be just friendly. A beautiful mind needs blessing with not only tenderness and richness but also a strong side that controls one’s feelings.
In our project, we will comprehensively evaluate Snow White story from the perspectives of two beauty, physical beauty and inner beauty.

2. Our goal is to evaluate Snow White Story in terms of real beauty.

“Magic Mirror on the wall, who is the fairest one of all?”

Once upon a time, there lived a Queen.
She was the fairest in the world and she herself also believed so.

Each time the Queen asked, “Magic Mirror on the wall, who is the fairest one of all?,” the mirror would give the same answer.
“You are the fairest one of all.”
This pleased the Queen greatly, for she knew that her Magic mirror could speak nothing but the truth and she asked it the same question.

“Magic Mirror on the wall, who is the fairest one of all?”

One winter night, the Queen asked her mirror as usual.
“Magic mirror on the wall, who is the fairest one of all?”
The mirror answered,
“Snow White is the fairest one.”

The Snow White was Queen’s daughter-in-law.
She was a kind and pure girl, and just turned seven years old.

Then the Queen was shocked, and beside herself with rage.
And thought,
“If I kill Snow White, I would be the fairest one of all again.”

The Queen prepared a poisoned apple.

She decided to transform into a Witch and give the apple to Snow White.
Snow White, who always takes people at their word , bit the apple, then sank into unconsciousness soon.

The Dwarfs found Snow White and they grieved her “death,” but they built a coffin and put her in it carefully.

One day, a Price from a neighboring country passed by the Dwarfs’ house.

Although he knew her death, he couldn’t help lifting her up because of her beauty.
Then, she opened her eyes!

This dislodged from Snow White’s throat the piece of poisoned apple that she had bitten off.


Awakened Snow White was adored by everyone and lived happily ever after.


THE END



The beauty is defined as follows in this project.

Beauty = physical beauty + inner beauty


In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP and GFP.
We use the graph shown below to evaluate. The vertical axis of the graph shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time.
No matter how beautiful one’s physical appearance is, the one cannot be a real beauty unless has a beautiful mind. We will show you which woman has a real beauty, Snow White or the Queen.

Fig. 2-2-1.


Even if we intended to evaluate the real beauty, we cannot evaluate it unless we represent the story. At the very beginning, we represent the story.

3. Introduction of system forming the basis

Before introduction of our genetic circuit’s design, we will introduce a system that form the basis of our project.
They are TA system.
   What is TA system?
   The art of MazF / MazE
The expression of real beauty by MazE / MazF
MazE / MazF expression of real beauty
The creation of real beauty under magic MazE / MazF
The creation of real beauty enchanted by MazE / MazF
The creation of real beauty under the spell of MazE / MazF



3.1 What is TA system?

Toxin-antitoxin system


A toxin-antitoxin system is composed of two or more cognate genes that encode toxins and antitoxins. Toxins are proteins, whereas antitoxins are either proteins or non-coding RNAs. [1] Many prokaryotes have toxin-antitoxin systems, and organisms often have them in multiple copies. [2][3] Changes in the physiological conditions, such as stress conditions or viral infection leads to the antitoxin degradation, and proteases degrade many antitoxins. Unleashed toxin proteins impede or alter cellular processes including translation, cell division, DNA replication, ATP synthesis, mRNA stability or cell wall synthesis and lead to dormancy. This dormant state probably enables the bacteria to survive in unfavorable conditions. In general, toxin is more stable than antitoxin, but antitoxin is expressed to a higher level.

一つ目のtoxin-anti-toxin : MazF / MazE


MazF is a toxin protein, and MazE is its cognate antitoxin protein. MazF is a ribosome-independent endoribonuclease. This endoribonuclease activity leads to bacterial growth arrest. MazE and MazF form homodimers, and MazF dimer cleaves mRNAs at ACA sites. [4] One MazE dimer binds to two MazF dimers, thereby inactivates endoribonuclease activity of MazF dimer. [5] MazE is labile, subjected to degradation by ClpAP protease, whereas MazF is more stable. [6]

二つ目のtoxin-anti-toxin : YafO / YafN


YafO is a toxin protein, and YafN is its cognate antitoxin protein. [7] YafO is a ribosome-associated mRNA interferase that cleaves mRNAs’ downstream 11-13 bases of the translation initiation sites. When 70S ribosome is dissociated, YafO associates with 50S ribosome subunit and gets endoribonuclease activity. YafO and YafN forms a complex Via binding with YafN, YafO is neutralized. [8] YafN is labile, subjected to degradation by Lon protease, whereas YafO is more stable [9].

The art of MazF / MazE

In our project, mazF and mazE are incorporated as a toxin and an antitoxin, respectively. Functioning of these two proteins makes it possible to evaluate the beauty of Snow White with GFP and RFP.
We will give you an example for a better understanding. Imagine that RFP is expressed little by little in E. coli.
When MazF expression is induced, the mRNA of GFP is cut, and GFP cannot be translated. Additionally, when MazE is induced, MazE and MazF forms a complex. MazF loses its function, which restarts GFP translation.

In this way, we can control protein translation. That’s why we decided to represent Snow White story.

4．Explanation of the circuit designed by us

We introduce the genetic circuits which we designed. Our project begins from the scene where the Magic Mirror answers the Queen’s question. We pick up 4 famous scenes containing this scene and will introduce genetic circuits in each scene in order.

   4.1　Scene1 : The magic mirror’s answer
   4.2　Scene2 : The Queen’s trap
   4.3　Scene3 : Snow White's sleep
   4.4　Scene4 : The Prince’s rescue



4.1.　Scene1 : The magic mirror’s answer


-----
“Magic mirror on the wall, who is the fairest one of all?”
The mirror answered,
“Snow White is the fairest one.”
-----


The story starts with the scene where it snows and gets cold. The magic Mirror coli can produce RhlI protein under low temperature condition. RhlI protein production leads to the production of the signaling molecule, C4HSL which tells the Queen that Snow White is the fairest, and the Queen coli receives this molecule.

4.2. Scene2 : The Queen’s trap


------
The Queen prepared a poisoned apple.
She decided to transform into a Witch and give the apple to Snow White.
------
4.2.1 reporter assay
First of all, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) when added 3 types of AHLs.



Although C12 is added to Plux, we found that the promoter activity can be seen. In addition, the graph shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when added C4.

The Queen, which has received C4HSL, produces LasI and MazF.LasI produces a signaling molecule, 3OC12HSL which is the Poisoned Apple. Additionally, produced MazF inhibits the translation in the Queen. If the translation is inhibited the moment MazF is expressed, sufficient amount of LasI will not be produced, and neither will 3OC12HSL. Then the Queen will fail the assassination of Snow White.

4.3. Scene3 : Snow White's sleep


-----
Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.
-----

The basic design of Snow White coli ’s genetic circuit is almost the same as the Queen coli’s. Snow White coli, which has received the C12HSL the Poisoned Apple, expresses hlI and MazF. Produced RhlI synthesizes signaling molecule C4HSL. As was described in the introduction of the Queen in the previous section, the Queen coli can receive C4HSL and monitor Snow White coli through this molecule. In addition, MazF expression inhibits the translation in Snow White coli.


4.4. Scene4 : The Prince’s rescue


-----
Although he knew her death, he couldn’t help lifting her up because of her beauty.
Then, she opened her eyes!
-----

The Prince coli which has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with more than 7 carbons. This leads to the degradation of the Poisoned Apple 3OC12HSL. When the Poisoned Apple is degraded, MazF expression is inhibited in Snow White, and gradually its function is counteracted by MazE. Then, translation restarts in Snow White coli.


5. Q.E.D. for the representation of Snow White

We conducted an experiment and simulated to confirm that the above four scenes can be represented. We introduced concepts in each scene and demonstrated them. Additionally, based on them, we simulated the representation of Snow White story.

>

5.1 Cold inducible promoter functions at 18°C in Scene 1
It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. This story never begins unless the Magic Mirror answers the Queen’s question.

The experiment was conducted with BBa_1949001. We cultivated for each sample at 18°C and 37°C and measured the GFP / Turbidity with a plate reader. The experimental result showed that samples cultured at 18°C had higher fluorescence intensity of GFP than those cultured at 37°C. Thus, we found that the story begins by lowering the culture temperature.

5.2 The screening of rhl promoter with optimal strength for Scene 2
The Queen coli gave the Poisoned Apple, and Snow White coli falls asleep. We use cell-cell communication to represent this scene, so we need the reporter with optimal strength to make the genetic circuit designed by us work properly. Therefore, we introduce rhl system assay as a concept in this Scene(校正お願いしたらもうちょい詳しくってコメントが来たのでお願いします…). We improved the function of rhl promoter through the process listed below and succeeded in newly obtaining of optimal rhl promoter mutants for our project.


5.2.2　rhlプロモーターの強度に関するシミュレーション
First of all, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) when added 3 types of AHLs.
The above diagram shows the intensity of the two promoters should be in the red region in the figure. The green point shows the relationships of the promoters which we intended to use primarily. In order to move to the red region, we found that it is needed to improve Prhl and increase its expression level.



5.2.3　Prhlの改良
We made mutants of rhl promoter by inserting a point mutation into the wild type (WT) rhl promoter. In the experiment, we added reagent AHLs into the reporters and examined their fluorescence intensities. As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl (NM).
iGEM Tokyo_Tech has Improved rhl promoter in the past. However, comparing the Prhl (NM) to Prhl (LR), the SN ratio of Prhl (NM) was higher than that of Prhl (LR). Futuremore, the result shows that Prhl (LR)has crosstalk with C12-AHL at a constant rate. When representing Snow White story, and the crosstalk to C12 happens, the Queen coli will suicide by eating the Poisoned Apple made by herself. Therefore, we made the mutants using WT as a template.