Part:BBa_K2599014
T7 Promoter+RBS+Leucocyclicin Q+intein+CBD
NCTU_Formosa 2018 designed a composite part encoding the Leucocyclicin Q sequence (BBa_K2599006), 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 Leucocyclicin Q
Introduction
Leucocyclicin Q, produced by Leuconostoc mesenteroides TK41401, possesses antimicrobial activity against broad range of bacteria srains, and particularly shows strong activity against Bacillus coagulans. Furthermore, it shows high stability against different pH value or heat stress.
The secondary structure prediction analysis of this bacteriocins revealed four identical α-helices, all having subtle amphiphilic characteristics that are thought to play an important role in their antimicrobial action. Also, Leucocyclicin Q was proved to be a cyclic bacteriocin in which N and C termini are bound to each other. In addition, a leucine residue constitutes the N terminus of cleaved leucocyclicin Q. Also, its structure shows 72% similarity to Lactocyclicin Q.
Mechanism of Leucocyclicin Q
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 bacteriocin activity was primarily recovered in the 0.5 M NaCl fraction of the cation-exchange chromatography.
Figure 2. Mechanism of bacteriocin
Features of Leucocyclicin Q
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 Leucocyclicin Q targets including Enterococcus faecalis, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC209 bactibase].
2. Eco-friendly
Since Leucocyclicin Q 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
Leucocyclicin Q 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.
Leucocyclicin Q is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 438.38.
Figure 3. The prediction result of Leucocyclicin Q.
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 Leucocyclicin Q sequence length is around 186 b.p. For the composite part, the sequence length should be near at 1230 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 Leucocyclicin Q fused with intein was induced by IPTG in E. coli , and intein-Leucocyclicin Q specifically bound to the column through chitin binding domain would be purified.
Figure 5. The mass of Leucocyclicin Q is around 34.4 kDa. No.1 is E. coli without transformed plasmid, No.2 is pTXB1 backbone without the target peptide, and No. 3-8 are Leucocyclicin Q
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 Leucocyclicin Q inhibiting ability throughout 4 hours.
Figure 7. Bar diagram that showed percentage resistance of Leucocyclicin Q to Bacillus subtilis 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 8. LB Agar plate of sterilization of Leucocyclicin Q+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 1094
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 817
Illegal AgeI site found at 907 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 737
Reference
1. Masuda, Y., et al. (2011). "Identification and characterization of leucocyclicin Q, a novel cyclic bacteriocin produced by Leuconostoc mesenteroides TK41401." Appl Environ Microbiol 77(22): 8164-8170.
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