Part:BBa_K2599012
T7 Promoter+RBS+Enterocin 96+intein+CBD
NCTU_Formosa 2018 designed a composite part encoding the Enterocin 96 sequence (BBa_K2599004), 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 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 by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The enterocin 96 sequence length is around 219 b.p. For the composite part, the sequence length should be near at 1263 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 96 fused with intein was induced by IPTG in E. coli , and intein-enterocin 96 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 96 + intein + CBD (BBa_K2599012, 35.9 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 96 inhibiting ability throughout 4 hours.
Figure 7. Bar diagram that showed percentage resistance of Enterocin 96 (46.47%) 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 96 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 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1127
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 850
Illegal AgeI site found at 940 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 770
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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