Part:BBa_K2599009

T7 Promoter+RBS+Bovicin HJ50+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Bovicin HJ50 sequence (BBa_K2599001), 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

Bovicin HJ50 is isolated from Streptococcus bovis HJ50. It contains a disulfide bridge and shows similarity to type AII lantibiotics. Like most of the bacteriocins produced by lactic acid bacteria, Bovicin HJ50 showed a narrow range of inhibiting activity.

Mechanism of Bovicin HJ50

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. Details are on our project page.

The bactericidal activity of Bovicin HJ50 is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores.

Features of Subtilosin

1. Species Specific

Bacteriocin target strains or closely related species. The organisims that Bovicin HJ50 targets including Bacillus megaterium, Bacillus subtilis, Bacillus coagulans, etc.

2. Eco-friendly

Since subtilosin is a polypeptide naturally produced by bacteria itself and can inhibit other bacteria without much environment impact. It don't pose threat to other organisms like farm animals or humans. Therefore, this toxin will not cause safety problem.

3. Biodegradable

Subtilosin is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.

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 1077
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 95
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 800
  Illegal AgeI site found at 890
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 720
  

Reference