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.

Since enterocin B belongs to class II bacteriocin, it is a small and heat stable peptide with a narrow spectrum of activity. Also, Enterocin A and enterocin B have very similar inhibitory spectra. They both 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.

Figure 2. Mechanism of bacteriocin

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.

Peptide Prediction

NCTU_Formosa 2017 had compeleted a [http://2017.igem.org/Team:NCTU_Formosa/Model peptide prediction model] that can predict peptide for new function. In this model, they used scoring card method (SCM) for machine learning. This year, NCTU_Formosa 2018 continued to use the same method for predicting antimicrobial peptide, in order to seek more candidates for our project.

Enterocin B is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 464.36.

Figure 3. The prediction result of Enterocin B.

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 4. 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 5. SDS-PAGE analysis. M: protein Ladder 5–245 kDa, C: negative control (only intein+CBD ,28 kDa), E: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa)

Inhibition Ability Analysis

To verify the fuction of bacteriocins, we target the major bacteria in soil, Bacillus subtilis. Positive control in the experiment is Ampicillin while the negative control is Bacillus subtilis without adding bacteriocins. We record record OD₆₀₀ values of samples with Elisa Reader. The growth curve of Bacillus subtilis can be observed in our resluts.

Figure 6. Normalized growth curve of Bacillus subtilis that showed Enterocin B inhibiting ability throughout 4 hours.

Figure 7. Bar diagram that showed percentage resistance of Enterocin B (46.06) to Bacillus subtilis after 4 hours.

Dose Response Assessment

We diluted samples into three different concentration, which is 0.5, 0.25 and 0.125 times of primitive samples. The positive control in this experiment is Ampicillin and the negative control is Bacillus subtilis without adding bacteriocins. All the data are triplicated and normalized.

Figure 8. Bar diagram that showed dose response of Enterocin B after 4 hours.

Safety

In the future, we are going to spray our bio-stimulator into the environment. To make sure whether the bacteria contain anti-microbial peptide will not exist in the final product, we design the processing standards in the laboratory.

Bacteriocins are usually heat stable, we use high-temperature sterilization to double make sure our peptide solution does not contain any living E. coli. However, peptides may degrades after long time sterilization. To find out the best fitted time for sterilization, we boiled our bacteriocins for 0, 15, 30, and 45 minutes, and put them on LB Agar plate and cultured it at 37℃ for 16 hours.

From the result of the plate, we can easily observe that bacteria exists only in the sample that is not boiled. After fifteen minutes of sterilization, there are no alive bacterias exist.

Figure 9. LB Agar plate of sterilization of Enterocin B+intein+CBD. (A)Negative control:LB broth. (B)Sterilize for 0 minutes. (C)Sterilize for 15 minutes. (D)Sterilize for 30 minutes. (E)Sterilize for 45 minutes.

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

1. Casaus, P., et al. (1997). "Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A." Microbiology 143(7): 2287-2294.

2. Franz, C. M., et al. (1999). "Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900." Appl Environ Microbiol 65(5): 2170-2178.