Difference between revisions of "Part:BBa K2904050"
(→‎Summary)
 
(64 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2904050 short</partinfo>
 
<partinfo>BBa_K2904050 short</partinfo>
 +
<br>
  
−
The sfGFP was regulated by the Adda riboswitch containing the original Adda riboswitch, Stabilizer and Tuner A.
+
==<strong>Design</strong>==
  
 +
===Background of 2019 OUC-China's project——RiboLego===
 +
<p>
 +
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.
 +
</p>
 +
 +
===The construction of this part===
 +
<p>
 +
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 <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 [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.
 +
<br>
 +
 +
==<strong>Result</strong>==
 +
We did two kinds of experiments to help us confirm the function of modular Adda riboswitch containing Tuner A.
 +
</p>
 +
===The result by confocal microscopy===
 +
 +
<p>
 +
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.
 +
[[Image:T--OUC-China--sjbb.png|center|thumb|500px|'''Figure1: The fluorescence images represent situation when fluorescence excitation by confocal microscopy. The images show E.coli with 2-AP. Compared with the original Adda riboswitch system and Adda fusion construct, an obvious fluorescence can be observed in modular Adda riboswitch system.'''  ]]
 +
<br>
 +
</p>
 +
 +
===The result by microplate reader===
 +
 +
<p>
 +
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.
 +
<br>
 +
</p>
 +
[[Image:T--OUC-China--050microplate.jpg|center|thumb|400px|'''Figure1: The results of modular Adda riboswitch containing Tuner A by microplate reader.'''  ]]
 +
 +
==<strong>Summary</strong>==
 +
 +
<p>
 +
This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation. To verify the functionality of different Tuners, we engineered different modular Adda riboswitches including Tuner A to E respectively. All circuits selected sfGFP as the target gene. Using different Tuners, muti-level regulation can be achieved.
 +
<br>
 +
[http://2019.igem.org/Team:OUC-China/Model The method we used to design different Tuners is on this page!]
 +
<br>
 +
First, microplate reader was used to measure the fluorescence intensity of sfGFP. Data is shown for each construct until steady state is reached (at least two consecutive subsequent data points do not increase fluorescence). The results demonstrate that these Tuners are capable of shifting and tuning the induction response of modular Adda riboswitches.
 +
<br>
 +
[[Image:T--OUC-China--addapoint.jpg|center|thumb|400px|'''Figure3: The results of modular Adda riboswitches containing different Tuners by microplate reader.'''  ]]
 +
<br>
 +
Then we also tested our modular Adda riboswitches by flow cytometer. The figure below shows the measured expression distributions at the same induction for modular Adda riboswitch with different Tuners.
 +
[[Image:T--OUC-China--flow.png|center|thumb|400px|'''Figure3: The results of modular Adda riboswitches containing different Tuners by flow cytometer. Tuner A:Red; Tuner B:Blue; Tuner C:Orange; Tuner D:Green; Tuner E:Dark Green.'''  ]]
 +
<br>
 +
By all the experiments mentioned before, we proved that Tuners work as expectations successfully. They are expected to serve as a powerful and tunable tool of riboswitch for future iGEM teams based on their demand. 
 +
<br>
 +
If you are interested in the other parts we designed, you can click modular riboswitches containing [https://parts.igem.org/Part:BBa_K2904051 Tuner B],[https://parts.igem.org/Part:BBa_K2904052 Tuner C],[https://parts.igem.org/Part:BBa_K2904053 Tuner D],[https://parts.igem.org/Part:BBa_K2904054 Tuner E]and [https://parts.igem.org/Part:BBa_K2904082 Tuner S].
 +
 +
</p>
 
<!-- 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 08:48, 21 October 2019


aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner A

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

We did two kinds of experiments to help us confirm the function of modular Adda riboswitch containing Tuner A.

The result by confocal microscopy

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.

Figure1: The fluorescence images represent situation when fluorescence excitation by confocal microscopy. The images show E.coli with 2-AP. Compared with the original Adda riboswitch system and Adda fusion construct, an obvious fluorescence can be observed in modular Adda riboswitch system.


The result by microplate reader

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.

Figure1: The results of modular Adda riboswitch containing Tuner A by microplate reader.

Summary

This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation. To verify the functionality of different Tuners, we engineered different modular Adda riboswitches including Tuner A to E respectively. All circuits selected sfGFP as the target gene. Using different Tuners, muti-level regulation can be achieved.
[http://2019.igem.org/Team:OUC-China/Model The method we used to design different Tuners is on this page!]
First, microplate reader was used to measure the fluorescence intensity of sfGFP. Data is shown for each construct until steady state is reached (at least two consecutive subsequent data points do not increase fluorescence). The results demonstrate that these Tuners are capable of shifting and tuning the induction response of modular Adda riboswitches.

Figure3: The results of modular Adda riboswitches containing different Tuners by microplate reader.


Then we also tested our modular Adda riboswitches by flow cytometer. The figure below shows the measured expression distributions at the same induction for modular Adda riboswitch with different Tuners.

Figure3: The results of modular Adda riboswitches containing different Tuners by flow cytometer. Tuner A:Red; Tuner B:Blue; Tuner C:Orange; Tuner D:Green; Tuner E:Dark Green.


By all the experiments mentioned before, we proved that Tuners work as expectations successfully. They are expected to serve as a powerful and tunable tool of riboswitch for future iGEM teams based on their demand.
If you are interested in the other parts we designed, you can click modular riboswitches containing Tuner B,Tuner C,Tuner D,Tuner Eand Tuner S.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 407