Difference between revisions of "Part:BBa K2904053"
 
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This part was used to validate our design principle of modular riboswitch. We employed activating Adda riboswitch, which can regulate the expression of <i>adenosine deaminase</i> by binding 2-aminopurine in Vibrio vulnificus.The first 150bp of <i>adenosine deaminase</i> was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used [https://parts.igem.org/Part:BBa_K2904110 sfGFP]as the reporter gene to reflect output of our system. Besides, [https://parts.igem.org/Part:BBa_K2904000 Tuner A] was inserted between Stabilizer and sfGFP. The modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner A was under control of [https://parts.igem.org/Part:BBa_K2904111 the tetracycline promoter], which was induced by aTc.  
 
This part was used to validate our design principle of modular riboswitch. We employed activating Adda riboswitch, which can regulate the expression of <i>adenosine deaminase</i> by binding 2-aminopurine in Vibrio vulnificus.The first 150bp of <i>adenosine deaminase</i> was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used [https://parts.igem.org/Part:BBa_K2904110 sfGFP]as the reporter gene to reflect output of our system. Besides, [https://parts.igem.org/Part:BBa_K2904000 Tuner A] was inserted between Stabilizer and sfGFP. The modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner A was under control of [https://parts.igem.org/Part:BBa_K2904111 the tetracycline promoter], which was induced by aTc.  
 
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==<strong>Result</strong>==
 
==<strong>Result</strong>==
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We did three kinds of experiments to help us confirm the function of modular Adda riboswitch containing Tuner A.
 
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===The result by confocal microscopy===
 
  
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===Modular Adda riboswitch===
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For the sake of functional test, other 2 circuits are set, Adda-sfGFP and Adda-Stabilizer-sfGFP, which also were under control of the same promoter. By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the adenine riboswitch had sfGFP introduced directly. The direct fusion of sfGFP to Stabilizer yielded very clear inclusion bodies, manifested as distinct spots present at one pole of the cell which are formed by misfolded insoluble proteins. By comparison, the modular Adda riboswitch yielded soluble working protein since Tuner has the ability to insulation the target gene from Stabilizer.  
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In order to validate the effect of Tuner D, we utilized Adda riboswitch, which can regulate the expression of <i>adenosine deaminase</i> by binding 2-aminopurine in <i>Vibrio vulnificus</i>. The first 150bp of <i>adenosine deaminase</i> was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used Tuner D to construct [https://parts.igem.org/Part:BBa_K2904011 modular Adda riboswitch] and [https://parts.igem.org/Part:BBa_K2904110 sfGFP]as the reporter gene to reflect output of our system.
 
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===The result by microplate reader===
 
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The qualitative experiment is not enough to analyze the modular Adda riboswitch. So we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that the modular Adda riboswitch can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.
 
 
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We tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that Tuner D can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.
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[[Image:T--OUC-China--A_banana.jpg|center|thumb|400px|'''Fig.1 The results of modular Adda riboswitch containing Tuner A by microplate reader.'''  ]]
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==<strong>Summary</strong>==
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This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation.  
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[http://2019.igem.org/Team:OUC-China/Model The method we used to design different Tuners is on this page!]
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The results demonstrate that these Tuners are capable of shifting and tuning the induction response of modular Adda riboswitches.
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[[Image:T--OUC-China--004micro.png|center|thumb|800px|'''Figure2: The results of modular Adda riboswitch containing Tuner D by microplate reader.''']]
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The above results demonstrate that Tuner D can help achieve tunable and efficient gene regulation. Besides, we also designed Tuner A to E.[http://2019.igem.org/Team:OUC-China/Model The design process about Tuners is on this page!]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:38, 21 October 2019


aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner D

Design

Background of 2019 OUC-China's project——RiboLego

Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a ‘plug and play' device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function.

The construction of this part

This part was used to validate our design principle of modular riboswitch. We employed activating Adda riboswitch, which can regulate the expression of adenosine deaminase by binding 2-aminopurine in Vibrio vulnificus.The first 150bp of adenosine deaminase was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used sfGFPas the reporter gene to reflect output of our system. Besides, Tuner A was inserted between Stabilizer and sfGFP. The modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner A was under control of the tetracycline promoter, which was induced by aTc.

Result

Modular Adda riboswitch

In order to validate the effect of Tuner D, we utilized Adda riboswitch, which can regulate the expression of adenosine deaminase by binding 2-aminopurine in Vibrio vulnificus. The first 150bp of adenosine deaminase was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used Tuner D to construct modular Adda riboswitch and sfGFPas the reporter gene to reflect output of our system.

We tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that Tuner D can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.

Figure2: The results of modular Adda riboswitch containing Tuner D by microplate reader.




The above results demonstrate that Tuner D can help achieve tunable and efficient gene regulation. Besides, we also designed Tuner A to E.[http://2019.igem.org/Team:OUC-China/Model The design process about Tuners is on this page!]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 358
    Illegal NgoMIV site found at 370
  • 1000
    COMPATIBLE WITH RFC[1000]