Difference between revisions of "Part:BBa K2904001"

(→‎The construction of this part)
(→‎Summary)
 
(12 intermediate revisions by the same user not shown)
Line 15: Line 15:
 
∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally.
 
∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally.
  
−
The following diagram shows the structure of Tuner A and we marked each region clearly.
+
The following diagram shows the structure of Tuner B and we marked each region clearly.
 
<br>
 
<br>
 
[[Image:T--OUC-China--tunerbstr.jpg|center|thumb|600px|'''Figure1: The structure of Tuner B.'''  ]]
 
[[Image:T--OUC-China--tunerbstr.jpg|center|thumb|600px|'''Figure1: The structure of Tuner B.'''  ]]
  
 
==<strong>Result</strong>==
 
==<strong>Result</strong>==
−
===Overview===
+
Using this part, we could engineer modular riboswitch. In order to validate the effect of Tuner B, 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 B 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.
−
Using this part, we could engineer many modular riboswitches. In order to validate the effect of Tuner A, we utilized four kinds of riboswitches, including Adda riboswitch, Btub riboswitch, cobalamin riboswitch and FourU. The results demonstrate that Tuner A can help achieve tunable and efficient gene regulation. Besides, we also used Tuner A to explore the source and length of Stabilizer. [http://2019.igem.org/Team:OUC-China/Results More results are on this page!]
+
 
<br>
 
<br>
 +
<br>
 +
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 B can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.
 +
<br>
 +
[[Image:T--OUC-China--051microplate.jpg|center|thumb|400px|'''Figure2: The results of modular Adda riboswitch containing Tuner B by microplate reader.'''  ]]
  
−
===Modular Adda riboswitch===
+
==<strong>Summary</strong>==
−
First, we employed 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 A to construct [https://parts.igem.org/Part:BBa_K2904010 modular Adda riboswitch] and [https://parts.igem.org/Part:BBa_K2904110 sfGFP]as the reporter gene to reflect output of our system.
+
 
 +
 
 +
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!]
 
<br>
 
<br>
 +
First, microplate reader was used to measure the fluorescence intensity of sfGFP. The figure below selects the data when 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>
 
<br>
−
For the sake of functional test, other 2 circuits are set, Adda-sfGFP and Adda-Stabilizer-sfGFP, which also were under control of the tetracycline 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 A has the ability to insulation the target gene from Stabilizer.  
+
[[Image:T--OUC-China--addapoint.jpg|center|thumb|400px|'''Figure3: The results of modular Adda riboswitches containing different Tuners by microplate reader.'''  ]]
 
<br>
 
<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|'''Figure4: 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>
 
<br>
−
The qualitative experiment is not enough to analyze the modular Adda riboswitch containing Tuner A. 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 Tuner A can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.
+
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>
 
<br>
−
[[Image:T--OUC-China--A_banana.jpg|center|thumb|400px|'''Fig.1 The results of modular Adda riboswitch containing Tuner A by microplate reader.'''  ]]
+
If you are interested in the other parts we designed, you can click modular riboswitches containing [https://parts.igem.org/Part:BBa_K2904050 Tuner A],[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_K2904055 Tuner S].
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:27, 20 October 2019


Tuner B

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

We defined a Tuner element to include a repressing region, a RBS region and a coupled junction region. The repressing region is the reverse complement of a subsequence of the RBS region so that Tuner can form a hairpin with appropriate ∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally. The following diagram shows the structure of Tuner B and we marked each region clearly.

Figure1: The structure of Tuner B.

Result

Using this part, we could engineer modular riboswitch. In order to validate the effect of Tuner B, 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 B 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 B 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 B 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. The figure below selects the data when 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.

Figure4: 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 A,Tuner C,Tuner D,Tuner Eand Tuner S.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]