Difference between revisions of "Part:BBa K3722006"

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===Biology===
 
===Biology===
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After linked the Fre to SttH, the total expression of SttH decreased, but all the chimeric enzymes showed highly increased ratios of their soluble forms in E. coli. The molar concentrations of soluble Fre-Linker–SttH was 1.64 μM, which was more than three times higher than that of SttH by itself[1].
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After linked the Fre to SttH, the total expression of SttH decreased, but all the chimeric enzymes showed highly increased ratios of their soluble forms in E. coli. The molar concentrations of soluble Fre-Linker–SttH was 1.64 μM, which was more than three times higher than that of SttH by itself<ref>Lee, J., Kim, J., Song, J.E. et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol 17, 104–112 (2021). https://doi.org/10.1038/s41589-020-00684-4</ref>.
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        <img src="https://2021.igem.org/wiki/images/0/0c/T--NWU-CHINA-A--part06.png" width="100%" style="float:center">
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===Usage===
 
===Usage===
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We connected Fre and SttH through the Linker to enhance the solubility of SttH. Then the ligation mixture was transformed into E. coli DH5α, which enabled the E. coli to express SttH protein. Due to the limited time, we did not characterize this part. As a result, there is a lack of data about this part.
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Here, we used <partinfo>BBa_K3722004</partinfo> and to construct the expression system and obtained the part <partinfo>BBa_K3722006</partinfo>, it can increase the solubility of SttH on our engineered bacteria. Due to the limited time, we only constructed part <partinfo>BBa_K3722009</partinfo> and <partinfo>BBa_K3722010</partinfo>, verified the protein expression by SDS PAGE analysis. As a result, there is a lack of quantized data about this part.
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        <img src="https://2021.igem.org/wiki/images/2/2b/T--NWU-CHINA-A--part01.png" width="100%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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===References===
 
===References===
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<references/>
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Latest revision as of 21:19, 21 October 2021


Fre-Linker-SttH

Construction of SttH and Fre fusion enzyme to enhance the solubility and activity of SttH.Fre is a unique N-terminal tag that efficiently enhanced SttH activity by increasing solubility as well as cofactor supply.


Biology

After linked the Fre to SttH, the total expression of SttH decreased, but all the chimeric enzymes showed highly increased ratios of their soluble forms in E. coli. The molar concentrations of soluble Fre-Linker–SttH was 1.64 μM, which was more than three times higher than that of SttH by itself[1].

Usage

Here, we used BBa_K3722004 and to construct the expression system and obtained the part BBa_K3722006, it can increase the solubility of SttH on our engineered bacteria. Due to the limited time, we only constructed part BBa_K3722009 and BBa_K3722010, verified the protein expression by SDS PAGE analysis. As a result, there is a lack of quantized data about this part.

References

  1. ↑ Lee, J., Kim, J., Song, J.E. et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol 17, 104–112 (2021). https://doi.org/10.1038/s41589-020-00684-4



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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 961
    Illegal NgoMIV site found at 1219
    Illegal AgeI site found at 1448
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 424