Composite

Part:BBa_K2904070

Designed by: Peng Deng   Group: iGEM19_OUC-China   (2019-10-15)
Revision as of 09:13, 21 October 2019 by Wawapeng (Talk | contribs)


sfGFP regulated by modular Adda riboswitch containing STA81 and 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

When it comes to Stabilizer, a necessary factor to consider is the length. We selected adenosine deaminase as Stabilizer of Adda riboswitch. Using docking matrix, we also selected four lengths of Stabilizer, called STA9, STA21, STA81 and STA129. Tuner A was inserted between Stabilizer and sfGFP. Before lab experiments, our program predicted that STA9 and STA21 could change the secondary structure of riboswitch because they all have high levels of punishment. In contrast, as the punishment of STA81 and STA129 both close to 1 ,they could stablize the secondary structure of riboswitch. This part consists of modular Adda riboswitch containing STA81 and sfGFP.

Result

We tested this part 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 containing STA81 can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.

File:T--OUC-China--070microplate.jpg
Figure1: The results of modular Adda riboswitch containing STA81 and Tuner A by microplate reader.

You can also observe the results about modular Adda riboswith containing STA129.


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 330


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