Team:Tianjin/Project

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==1. AegiSafe O-Key: The orthogonal system of regulating translation==
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The synthesis of protein relies on the transcription-translation network. In transcription, mRNA is synthesized through complementary base pairing by RNA polymerase from the DNA template, and is followed by translation. Translation is the third stage of protein biosynthesis. In translation, mRNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein. Therefore, we can say that translation is one of most important of activites in a cell. <br\>
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Due to its importance of protein synthesis, there are intricate and delicate regulation systems. To regulate transcription, cells alter the gene expression levels. This is called transcriptional regulation. Many means of transcriptional regulation, such as various mechanisms as specificity factors, activators, etc., are presented in a cell. There are also inducible and repressible systems, and transcription factor that can determine the initiation rate of transcription.<br\>
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<p class="menu_head">Project Contents</p>
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<a href="https://2012.igem.org/Team:Tianjin/Project/OrthogonalSystem">Orthogonal System</a>
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<a href="https://2012.igem.org/Team:Tianjin/Project/Regulation">Logic Metabolism Regulation</a>
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<a href="https://2012.igem.org/Team:Tianjin/Project/Gene">Future Work</a>
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<a href="https://2012.igem.org/Team:Tianjin/Data">BioBrick</a>
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<a href="https://2012.igem.org/Team:Tianjin/Project/Technology">Technology</a>
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<p class="menu_head">In this page</p>
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<a href="#Orthogonal_System">Orthogonal System</a>
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<a href="#Gene_Pollution_Prevention_and_Gene_Encryption">Safety Encryption</a>
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<a href="#Logic_Metabolism_Regulation">Logic Metabolism Regulation</a>
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While a number of genetic tools exist for regulating transcription in cells, far fewer tools exist for translation. Of the tools available in bacteria, the most popular are riboregulators, both cis- and trans-activating, and orthogonal ribosomes (o-ribosomes). In terms of reprograming translation, o-ribosomes are especially powerful as they enable one to partially decouple translation from the native protein synthesis machinery. In particular, o-ribosomes can translate genes with altered Shine-Dalgarno (SD) sequences not recognized by host ribosomes. Therefore, o-ribosomes can be used to explore gene expression dynamics as they potentially provide a method for tuning translation rates. Furthermore, o-ribosomes may have application in synthetic biology as they introduce new functionality within cells.<br\>
 
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In this year's project, we focus on modifying the Sine-Dalgarno (SD) sequence of the mRNA and the anti-Shine-Dalgarno (ASD) sequence of the 16S rRNA of the ribosome to construct a orthogonal translation system called the AegiSafe O-Key. Aegis was originally the shield that was associated with Zeus and Athena, which offers protection. AegiSafe means our orthogonal system secure the environment from potential biosafety issue. O-Key stands for Orthogonal Key, which indicates that our system can not only shut down contamination, but also using the orthogonal system to selectively open our translation pathway like a key. In the following sections, we will elaborate on the AegiSafe O-Key.<br\>
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<center><span style="font-size:46px;font-family:Cambria;margin-top:10px;line-height:80%">Project</span></center>
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<br>
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The Glossary:
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* The O-Key System -- Any orthogonal system containing a pair of orthogonal ribosome and mRNA
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* The O-Key -- the orthogonal ribosome, which serves like a key to translate the orthogonal mRNA
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* The O-Lock -- the orthogonal mRNA, which can only be deciphered by the O-Key.
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=Orthogonal System=
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[[file:TJU2012-Proj-fig-1.png|thumb|200px|right|'''Figure 1.''' Word "RNA" (from "http://www.mfpl.ac.at")]]
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By rationally mutate the Shine-Dalgarno (SD) and anti-Shine-Dalgarno (ASD) sequence, we are able to take advantage of the interaction of mRNA and ribosome to build our O-Key System of orthogonal ribosome and orthogonal mRNA. Within this system, we constructed an operon containing RFP and GFP coding sequence to verify the orthogonality of the O-Key. By selectively mutate the SD sequence of RFP or GFP, we were able to establish four translation pathways to characterize the effect of the O-Key System. In addition, we set up a model to predict the output of GFP and RFP under various circumstances. The model calculates the ΔG of ASD and SD sequence binding, and make use of this energy to evaluate the feasibility and translation efficiency of our O-Key System. The model turned out to be highly convincing as it corresponds with our wet lab result. In the end, both the wet and dry lab results matches our design. 
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=<span style="line-height:100%">Genetic Pollution Prevention and Genetic Encryption</span>=
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[[file:TJU2012-Proj-fig-2.png|thumb|400px|right|'''Figure 2.''' Comic of genetic pollution defence (from TJU iGEM　Team 2012)]]Aiming at preventing genetic pollution, we employed the O-Key System to establish a translational fence that can restrain unwanted protein expression. The convenience and effectiveness of the O-Key System will make it applied to a larger scale in genetic engineering. We predict different companies will embed the O-Key system in their various product to ensure biosafety. In the meantime, because the O-Key System includes a key and a lock, we can make use of this mechanism to encrypt information into cell or locking the product information. This characteristic showed a promising application in information encryption, intellectual property protection, etc. Furthermore, the O-Key System can be applied to the entire organism to construct an orthogonal organism. We began with the simplest creature - the phage, and worked on the RBS of its various protein. After mutation, the phage becomes a brand new orthogonal organism that can only infect the cells with orthogonal ribosomes.  Using this O-Key Phage, we greatly reduce risk of phage pollution in the lab, while performing regular experiment using the phage. At last, a successful interdisciplinary model that combines marketing and bioengineering was constructed to predict the diffusion of exogenous gene across space and time. This creative model used the analogy of human society and bacteria colony to predict the speed and probability of genetic transfer.
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=Logic Metabolism Regulation=
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[[file:TJU2012-Proj-fig-3.png|thumb|300px|right|'''Figure 3.''' Metabolism Network (from TJU iGEM　Team 2012)]]
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In this section, we describe the principles of Yeast Assembler, a novel way of assemble multiple fragments into a long operon, and specifically used this method to construct the gene needed to produce Violacein. The pathway of expressing violacein consists of five genes, and they build up a long operon. The conventional assembly methods for violacein takes too much time and labor, up to several weeks and offer resulting in failure, but using Yeast Assembler we can complete the whole process in a week. We will introduce and elaborate on the assembler in details. Through such an experiment, we could also prove the feasibilities of the O-Key System in regulating metabolism. Furthermore, we talked about the application of AND gate based on O-Key System in adjusting metabolism.
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Latest revision as of 16:00, 26 October 2012


Project


The Glossary:

  • The O-Key System -- Any orthogonal system containing a pair of orthogonal ribosome and mRNA
  • The O-Key -- the orthogonal ribosome, which serves like a key to translate the orthogonal mRNA
  • The O-Lock -- the orthogonal mRNA, which can only be deciphered by the O-Key.

Orthogonal System

Figure 1. Word "RNA" (from "http://www.mfpl.ac.at")

By rationally mutate the Shine-Dalgarno (SD) and anti-Shine-Dalgarno (ASD) sequence, we are able to take advantage of the interaction of mRNA and ribosome to build our O-Key System of orthogonal ribosome and orthogonal mRNA. Within this system, we constructed an operon containing RFP and GFP coding sequence to verify the orthogonality of the O-Key. By selectively mutate the SD sequence of RFP or GFP, we were able to establish four translation pathways to characterize the effect of the O-Key System. In addition, we set up a model to predict the output of GFP and RFP under various circumstances. The model calculates the ΔG of ASD and SD sequence binding, and make use of this energy to evaluate the feasibility and translation efficiency of our O-Key System. The model turned out to be highly convincing as it corresponds with our wet lab result. In the end, both the wet and dry lab results matches our design.

Genetic Pollution Prevention and Genetic Encryption

Figure 2. Comic of genetic pollution defence (from TJU iGEM　Team 2012)
Aiming at preventing genetic pollution, we employed the O-Key System to establish a translational fence that can restrain unwanted protein expression. The convenience and effectiveness of the O-Key System will make it applied to a larger scale in genetic engineering. We predict different companies will embed the O-Key system in their various product to ensure biosafety. In the meantime, because the O-Key System includes a key and a lock, we can make use of this mechanism to encrypt information into cell or locking the product information. This characteristic showed a promising application in information encryption, intellectual property protection, etc. Furthermore, the O-Key System can be applied to the entire organism to construct an orthogonal organism. We began with the simplest creature - the phage, and worked on the RBS of its various protein. After mutation, the phage becomes a brand new orthogonal organism that can only infect the cells with orthogonal ribosomes. Using this O-Key Phage, we greatly reduce risk of phage pollution in the lab, while performing regular experiment using the phage. At last, a successful interdisciplinary model that combines marketing and bioengineering was constructed to predict the diffusion of exogenous gene across space and time. This creative model used the analogy of human society and bacteria colony to predict the speed and probability of genetic transfer.

Logic Metabolism Regulation

Figure 3. Metabolism Network (from TJU iGEM　Team 2012)

In this section, we describe the principles of Yeast Assembler, a novel way of assemble multiple fragments into a long operon, and specifically used this method to construct the gene needed to produce Violacein. The pathway of expressing violacein consists of five genes, and they build up a long operon. The conventional assembly methods for violacein takes too much time and labor, up to several weeks and offer resulting in failure, but using Yeast Assembler we can complete the whole process in a week. We will introduce and elaborate on the assembler in details. Through such an experiment, we could also prove the feasibilities of the O-Key System in regulating metabolism. Furthermore, we talked about the application of AND gate based on O-Key System in adjusting metabolism.

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