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Composite

Part:BBa_K2599008

Designed by: YEN-LING CHEN   Group: iGEM18_NCTU_Formosa   (2018-09-16)
Revision as of 08:36, 18 September 2018 by Yen-ling (Talk | contribs)


T7 Promoter+RBS+Subtilosin+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Subtilosin sequence (BBa_K2599000), and then combined with a T7 promoter (BBa_I712074), a ribosome binding site (BBa_B0034), intein and chintin binding domain (CBD). Further information of our peptide can be found on our design page.


Figure 1 biobrick picture


Introduction

Bacillus subtilis produces a bacteriocin called Subtilosin that possesses antibacterial activity against certain gram-positive bacteria. The bacteriocins are a group of anitmicrobial peptides that are often distinguished from traditioinal antibiotics by their narrow range of avtivity against closely related bacteria.


Mechanism of Subtilosin

The bacteriocins inhibit their target organisms through pore formation. Though the mechanism of each inhibition is vary from species to species, the general process is conserved. The details is on our design page.

According to the reference, Subtilosin acts by fully depleting the transmembrane pH gradient (ΔpH) and causing an immediate efflux of intracellular ATP, but has no effect on the transmembrane electric potential. In addition, the paper also shows that membrane permeabilization occurred at concentrations of subtilosin that were significantly higher than the MIC level of E. coli tested strains.


Features of Subtilosin


Experiment Result

Cloning

We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Subtilosin sequence length is around 147 b.p. For the composite part, the sequence length should be near at 1191 b.p.


Figure 2 PCR


Expressing

We chose E. coli 2566 strain to express our antibacterial peptides. The expression of Subtilosin fused with intein was induced by IPTG in E. coli , and intein-subtilosin specifically bound to the column through chitin binding domain would be purified.


Figure 3 SDS


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1055
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 778
    Illegal AgeI site found at 868
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 698


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