Composite

Part:BBa_K3739038

Designed by: Ziyan Han   Group: iGEM21_XMU-China   (2021-09-09)
Revision as of 21:48, 21 October 2021 by YWK (Talk | contribs) (→‎Characterization)


J23100-B0030-Aly01-his-GFP-B0010

This part contains a Vibrio natriegens-optimized GFP coding sequence, and the GFP is fused at N-terminal with his-tag in order to let us purify the protein. His-GFP is fused with secretory peptide from Aly01.We use BBa_K3739038 to construct the expression system and help prove the function of some signal peptide.

Biology

Aly01 Aly01 is alginate lyase from Vibrio natriegens SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in E. coli. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.

GFP Protein tags are peptides genetically fused to the proteins of interest. There are many different kinds of tags developed for for different purposes. For labeling experiments, normally the proteins of interest are tagged directly by fluorescent tags. Green fluorescent protein (GFP) make it easier to study protein localization, interactions and dynamics when fusing with other peptides and proteins.

Usage

Here, we used BBa_K3739007 to construct the expression system and obtained the composite part BBa_K3739038. We transformed the constructed plasmid on the expression vector pET-28a (+) into V. natriegens to extract the protein. This protein work together with those from BBa_K3739041 and BBa_K3739043 to verify the function of our secretory peptides Aly01 and LMT.

Characterization

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Reference

1.Meng, Q. et al. Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase. Journal of the science of food and agriculture 101, 5154-5162, doi:10.1002/jsfa.11161 (2021).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 137
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 215


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