Part:BBa_K2599011

T7 Promoter+RBS+Enterocin B+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Enterocin B sequence (BBa_K2599003), 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 Enterocin B

Introduction

Same as enterocin A, enterocin B is also from Enterococcus faecium that contains disulfide bridge. However, enterocin B does not belong to the pediocin family of bacteriocins. The primary translation product was a 71 aa peptide containing a leader peptide, and it is found to be a heat-stable bacteriocins, which can synergistically act with enterocin A.

Mechanism of Enterocin B

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.

Enterocin A and enterocin B have very similar inhibitory spectra. It interact with anionic lipids that are abundantly present in the membranes of Gram-positive bacteria. And according to the reference, enterocin B is inhibitory to some strains that are not sensitive to enterocin A. Furthermore, enterocin B is able to kill the bacteria that survive from the enterocin A treatment.

Features of Enterocin B

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 B targets including Enterococcus faecalis, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC101 bactibase].

2. Eco-friendly

Since enterocin B 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

Enterocin B 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 enterocin B sequence length is around 210 b.p. For the composite part, the sequence length should be near at 1254 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 enterocin B fused with intein was induced by IPTG in E. coli , and intein-enterocin B 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 1118
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 841
  Illegal AgeI site found at 931
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 761
  

Reference