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 lac operator (K1624002), a ribosome binding site (BBa_B0034), intein and chintin binding domain (CBD) (BBa_K1465230). Further information of our peptide can be found on our design page.

Figure 1. Composite part of Bovicin HJ50

Introduction

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

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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. To see more details, please search for 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. Its pore-forming activity is significantly different from other lantibiotics, suggesting a novel antimicrobial mechanism.

Features of Bovicin HJ50

1. Species Specific

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

Thus this bacteriocin is one of the peptide candidates for our project, that can solve the unbalance microbiota of agriculture in Taiwan.

More target organisms can be found on [http://bactibase.hammamilab.org/BAC156 bactibase].

2. Eco-friendly

Since Bovicin HJ50 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.

top:16px;font-size:16px">3. Biodegradable</p>

Bovicin HJ50 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 by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The Bovicin HJ50 sequence length is around 171 b.p. For the composite part, the sequence length should be near at 1215 b.p. There are also some restrictioin sites at the two sides of our target protein, provided for future team to utilize the intein tag.

Figure 2. PCR product

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-bovicin HJ50 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