Part:BBa_K2599010

T7 Promoter+RBS+Enterocin A+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Enterocin A sequence (BBa_K2599002), 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). Further information of our peptide can be found on our design page.

Figure 1 biobrick picture

Introduction

Mechanism of Enterocin A

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 are antimicrobial peptides that will kill or inhibit bcterial strains closely related or non-related to produced bacteria, but will not harm the bacteria themselves by specific immunity proteins. The organisims that Enterocin A targets including Enterococcus faecalis, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC088]

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

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.

3. Biodegradable

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 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.

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-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