Part:BBa_K2599015

T7 Promoter+RBS+Durancin TW-49M+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Durancin TW-49M sequence (BBa_K2599007), 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. Composite part of Durancin TW-49M

Introduction

Durancin TW-49M produced by Enterococcus durans QU 49, is a nonpediocin-like class II bacteriocin. Enterococcus are highly competitive due to their resistance to wide range of pH and temperature. Therefore, the bacteriocins they produce shows their wide range effectiveness on pathogenic bacteria.

Mechanism of Durancin TW-49M

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.

Features of Durancin TW-49M

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 Lacticin Z targets including Enterococcus faecalis, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC159 bactibase].

2. Eco-friendly

Since Durancin TW-49M 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

Durancin TW-49M 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 Durancin TW-49M sequence length is around 213 b.p. For the composite part, the sequence length should be near at 1257 b.p.

Figure 2 PCR

Expressing

We chose E. coli 2566 strain to express our antibacterial peptides. The expression of Durancin TW-49M 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 1121
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 844
  Illegal AgeI site found at 934
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 764
  

Reference