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.

File:T--NCTU Formosa--Ent A 2.png

Figure 1. P_T7+RBS+Enterocin A+intein+CBD

Introduction

Bacteriocin from Enterococcus faecium, called enteriocin A, belongs to class IIa. These kind of bacteriocins do not have post-translational modifications. It is a small, heat-stable peptide that is known as pediocin-like bacteriocins, which may contain one or two disulfide bridges.

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.

Enterocin A, like most of the class IIa bacteriocins, acts on the cytoplasmic membrane of gram-positive cells. It will bind to the receptor leading to an irreversible opening of an interinsic channel, allowing to form a pore, and therefore dissipate the transmembrane elctrical potential.

Features of Enterocin A

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 bactibase].

2. Eco-friendly

Since enterocin A 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 A 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 enterocin A sequence length is around 192 b.p. For the composite part, the sequence length should be near at 1236 b.p.

Figure 2 PCR

Expressing

We chose E. coli 2566 strain to express our antibacterial peptides. The expression of enterocin A fused with intein was induced by IPTG in E. coli , and intein-enterocin A 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 1100
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 823
  Illegal AgeI site found at 913
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 743
  

Reference