Difference between revisions of "Part:BBa K2904053"
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<partinfo>BBa_K2904053 short</partinfo> | <partinfo>BBa_K2904053 short</partinfo> | ||
| − | sfGFP | + | ==<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 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. | ||
| + | <br> | ||
| + | ==<strong>Result</strong>== | ||
| + | |||
| + | ===Modular Adda riboswitch=== | ||
| + | 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. | ||
| + | <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 D can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period. | ||
| + | <br> | ||
| + | [[Image:T--OUC-China--004micro.png|center|thumb|800px|'''Figure2: The results of modular Adda riboswitch containing Tuner D by microplate reader.''']] | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | 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!] | ||
| + | <br> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 358
Illegal NgoMIV site found at 370 - 1000COMPATIBLE WITH RFC[1000]