Part:BBa_K2599004
Antimicrobial peptide - Enterocin 96
This biobrick is the basic part of the enterocin 96, more information please look for the composite part (BBa_K2599012).
Figure 1. Basic 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.
Figure 2. Mechanism of bacteriocin
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.
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 96 is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 464.06.
Figure 3. The prediction result of Enterocin 96.
Experiment Result
Cloning
We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Enterocin 96 sequence length is around 219 b.p. and the length of PCR product should be around 269 b.p.
Figure 4.Agarose gel electrophoretic pattern of Taq PCR product.
More experiment results of Enterocin 96, please look for the composite part BBa_K2599012
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 Enterocin 96+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. Izquierdo, E., et al. (2009). "Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese." Appl Environ Microbiol 75(13): 4273-4276.
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