Part:BBa_K2599012

T7 Promoter+RBS+Enterocin 96+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Enterocin 96 sequence (BBa_K2599004), 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 96

Introduction

Enterocin 96, a classII bacteriocin synthesized with a leader peptide, is produced by Enterococcus faecalis. It has strong activity against most gram-positive strains but almost no activity against gram-negative strains, such as Escherichia coli. The antimicrobial spectrum is relatively wide compared with other bacteriocins from lactic acid bacteria.

Mechanism of Enterocin 96

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 96, 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 96

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 96 targets including Enterococcus faecalis, Bacillus subtilis, Listeria monocytogenes, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC149 bactibase].

2. Eco-friendly

Since enterocin 96 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 96 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 96 sequence length is around 219 b.p. For the composite part, the sequence length should be near at 1263 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 96 fused with intein was induced by IPTG in E. coli , and intein-enterocin 96 specifically bound to the column through chitin binding domain would be purified.

Figure 3. The mass of enterocin 96 is around 35.9 kDa. No.1 is E. coli without transformed plasmid, No.2 is pTXB1 backbone without the target peptide, and No. 3-5 are enterocin 96.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1127
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 850
  Illegal AgeI site found at 940
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 770
  

Reference