Part:BBa_K2599007
Antimicrobial peptide - Durancin TW-49M
This biobrick is the basic part of the Durancin TW-49M, more information please look for the composite part (BBa_K2599015).
Figure 1. Basic part of Durancin TW-49M
Introduction
Durancin TW-49M produced by Enterococcus durans QU 49, is a nonpediocin-like class II bacteriocin without modified amino acid. 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.
Although there is an intramolecular disulfide bond in durancin TW-49M, it is not crucial for activity, while important for the stability of activity.
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.
Figure 2. Mechanism of bacteriocin
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.
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.
Durancin TW-49M is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 478.97.
Figure 3. The prediction result of Durancin TW-49M.
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. and the length of PCR product should be around 263 b.p.
Figure 4. Agarose gel electrophoretic pattern of Taq PCR product.
More experiment results of Bovicin HJ50, please look for the composite part BBa_K2599015.
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 5. LB Agar plate of sterilization of Durancin+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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
1. Hanchi, H., et al. (2018). "The Genus Enterococcus: Between Probiotic Potential and Safety Concerns—An Update." 9(1791).
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