Part:BBa_K2599009

T7 Promoter+RBS+Bovicin HJ50+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Bovicin HJ50 sequence (BBa_K2599001), 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 Bovicin HJ50

Introduction

Bovicin HJ50 is isolated from Streptococcus bovis HJ50. It shows similarity to type IIa lantibiotics, the largest group of lantibiotics. It comprises a linear N-terminal region and a globular C-terminal region. Its disulfide bond is especially essential for antimicrobial activity. Like most of the bacteriocins produced by lactic acid bacteria, Bovicin HJ50 showed a narrow range of inhibiting activity. Its antimicrobial activity has been proved in reference.

Mechanism of Bovicin HJ50

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.

The bactericidal activity of Bovicin HJ50 is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. Its pore-forming activity is significantly different from other lantibiotics, suggesting a novel antimicrobial mechanism.

Figure 2. Mechanism of bacteriocin

Features of Bovicin HJ50

1. Species Specific

Bacteriocins target strains or closely related species. The organisims that Bovicin HJ50 targets including Bacillus megaterium, Bacillus subtilis, Bacillus coagulans, etc.

Thus this bacteriocin is one of the peptide candidates for our project, that can solve the unbalance microbiota of agriculture in Taiwan.

More target organisms can be found on [http://bactibase.hammamilab.org/BAC156 bactibase].

2. Eco-friendly

Since Bovicin HJ50 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

Bovicin HJ50 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.

Bovicin HJ50 is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 459.87.

Figure 3. The prediction result of Bovicin HJ50.

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 Bovicin HJ50 sequence length is around 171 b.p. For the composite part, the sequence length should be near at 1215 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 Subtilosin fused with intein was induced by IPTG in E. coli , and intein-bovicin HJ50 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: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa)

Inhibition Ability Test

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 Bovicin HJ50 inhibiting ability throughout 4 hours.

Figure 7. Bar diagram that showed percentage resistance of Bovicin HJ50 (82.05%) 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 Bovicin HJ50 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 Bovicin HJ50+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 1077
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 95
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 800
  Illegal AgeI site found at 890
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 720
  

Reference

1. Liu, G., et al. (2009). "Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50." Microbiology 155(Pt 2): 584-593.

2. Xiao, H., et al. (2004). "Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50." Microbiology 150(Pt 1): 103-108.

3. Zhang, J., et al. (2014). "Type AII lantibiotic bovicin HJ50 with a rare disulfide bond: structure, structure-activity relationships and mode of action." Biochem J 461(3): 497-508.