Composite

Part:BBa_K3739041

Designed by: Ziyan Han   Group: iGEM21_XMU-China   (2021-09-09)
Revision as of 21:47, 21 October 2021 by YWK (Talk | contribs) (→‎Characterization)


J23100-B0030-LMT-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 LMT.We use BBa_K3739041 to construct the expression system and help prove the function of some signal peptide.


Biology

LMT
Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kind of LMTs existing in E.coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of Vibrio natriegens.
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_K3739010 to construct the expression system and obtained the composite part BBa_K3739041. 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_K3739038 and BBa_K3739043 to verify the function of our secretory peptides Aly01 and LMT.

Characterization

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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 NgoMIV site found at 661
    Illegal NgoMIV site found at 1097
    Illegal NgoMIV site found at 1832
    Illegal NgoMIV site found at 2068
    Illegal AgeI site found at 407
    Illegal AgeI site found at 497
    Illegal AgeI site found at 734
    Illegal AgeI site found at 1561
    Illegal AgeI site found at 2057
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None