After producing our target protein---bacteriocin, we investigate function of bacteriocins including Inhibition Zone and Inhibition Ability. We choose Bacillus subtilis as our target model.
Team:NCTU Formosa/Wet Lab/Functional Analysis
Inhibition Zone
To test the inhibition ability of bacteriocins in solid medium, we use Inhibition Zone to measure the effective inhibition of bacteriocins. We use the condensed culture spread to the agar plate and add bacteriocins and let it to dry. In that case, the dose of inhibition zone is as same as microdilution which can be in contrast with liquid condition.
Inhibition Ability Analysis
The purpose of this experiment is to verify that bacteriocins have function. Firstly, we cultivated Bacillus subtilis as bacteriocins’ inhibiting target. Centrifuge products which have been sonicated and purified them as samples. The positive control in this experiment is ampicillin and the negative control is Bacillus subtilis without adding bacteriocins. We use Elisa Reader to record O.D.600nm values of samples once an hour, if O.D.600 values decrease, it means that our bacteriocin inhibit the growth of Bacillus subtilis. Further, we can also know when does bacteriocin degrade by observing the growth curve of Bacillus subtilis. The pre-test data are all triplicated and normalized.
Inhibition Analysis of unpurified bacteriocins
After purifying bacteriocins, we do the inhibition analysis of unpurified bacteriocins, which only been sonicated after expression, and we find out that bacteriocins also have strong inhibition ability to Bacillus subtilis. Therefore, we do a series of experiments using unpurified bacteriocins, and the following data shows that each bacteriocin can have effective function without purifying, keeping costs of using bacteriocins as a biostimulator low.
Figure 6：Bar diagram that showed percentage resistance of six bacteriocins to Bacillus subtilis after four hours.
Lacticin Z(96.64%), Bovicin HJ50(82.05%), Enterocin 96(46.47%), Enterocin B(46.06%), Leucocyclicin Q(40.58%),Durancin TW-49M(19.52%).
Figure 7：Normalized growth curve of bacteriocins and Negative Control.
- Figure 6 shows six bacteriocins’ inhibiting ability to B. sub, and Durancin TW-49M has the most effective function, inhibiting 80% of B. sub’s growth.
- Figure 6 shows how O.D.600nm value of each sample decreases, indicating the strongest bacteriocin is Durancin TW-49M. It has strong inhibiting ability and its standard difference is also small, which means Durancin TW-49M is both effective and stable.
- The following data showed that each bacteriocin can have effective function without purifying, keeping costs of using bacteriocins as a bio-stimulator low.
Durancin TW-49M
Figure 3：Bar diagram that showed percentage resistance of Durancin TW-49M to Bacillus subtilis after acting for four hours. Durancin TW-49M (18.94%)
Figure 4：Inhibition Zone Test of Durancin TW-49M. A: Durancin TW-49M; B: Negative Control
Figure 5：Normalized growth curve of Bacillus subtilis that showed Durancin TW-49M inhibiting ability throughout four hours.
Enterocin B
Figure 6：Bar diagram that showed percentage resistance of Enterocin B to Bacillus subtilis after acting for four hours.
Enterocin B(44.24%)
Figure 7：Inhibition Zone Test of Enterocin B
A: Enterocin B; B: Negative Control
Figure 8：Normalized growth curve of Bacillus subtilis that showed Enterocin B inhibiting ability throughout four hours.
Enterocin 96
Figure 12：Bar diagram that showed percentage resistance of Enterocin 96 to Bacillus subtilis after acting for four hours.
Enterocin 96 (45.16%)
Figure 13：Inhibition Zone Test of Enterocin 96.
A: Enterocin 96
Figure 14：Normalized growth curve of Bacillus subtilis that showed Enterocin 96 inhibiting ability throughout four hours.
Bovicin HJ50
Figure 15：Bar diagram that showed percentage resistance of Bovicin HJ50 to Bacillus subtilis after four hours.
Bovicin HJ50 (82.05%)
Figure 16：Inhibition Zone Test of Enterocin 96.
A: Enterocin 96
Figure 17：Normalized growth curve of Bacillus subtilis that showed Bovicin HJ50 inhibiting ability throughout four hours.
Leucocyclicin Q
Figure 18：Bar diagram that showed percentage resistance of Leucocyclicin Q to Bacillus subtilis after four hours.
Leucocyclicin Q (40.58%)
Figure 19：Inhibition Zone Test of Enterocin 96.
A: Enterocin 96
Figure 20：Normalized growth curve of Bacillus subtilis that showed Leucocyclicin Q inhibiting ability throughout four hours.
Dose Response Assessment
Secondly, we diluted samples into three different concentration, which is 0.5, 0.25 and 0.125 times of primitive samples. Dose Response Assessment showed that the inhibition wasn’t caused by endotoxin. The positive control in this experiment is Ampicillin and the negative control is Bacillus subtilis without adding bacteriocins. The Dose Response Assessment data are all triplicated and normalized.
Figure 21：Bar diagram that showed dose response of Enterocin 96 after four hours.
Figure 22：Bar diagram that showed dose response of Leucocyclicin Q after four hours.
Figure 23：Bar diagram that showed dose response of Enterocin B after four hours.
Figure 24：Bar diagram that showed dose response of Bovicin HJ50 after four hours.
Figure 25：Bar diagram that showed dose response of Durancin TW-49M after four hours.
Conclusion
From our experiments we conclude that our 6 bacteriocins are functional. We began by performing both functional test in 96-well plate and inhibition zones. Our results indicate successful inhibition of Bacillus subtilis. Then, we moved on to dose response assessment. Of our 6 peptides, four show convincing results, following the trend of greater inhibition with higher dosage.
References
1. Aymerich, T., et al. (1996). "Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins." Appl Environ Microbiol 62(5): 1676-1682.
2. Hu, C.-B., et al. (2010). "Enterocin X, a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides." Applied and Environmental Microbiology 76(13): 4542-4545.
3. Iwatani, S., et al. (2007). "Characterization and Structure Analysis of a Novel Bacteriocin, Lacticin Z, Produced by Lactococcus lactis QU 14." Bioscience, Biotechnology, and Biochemistry 71(8): 1984-1992.
4. 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.
5. Liu, G., et al. (2009). "Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50." Microbiology 155(Pt 2): 584-593.
6. 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.
7. O'Keeffe, T., et al. (1999). "Characterization and heterologous expression of the genes encoding enterocin a production, immunity, and regulation in Enterococcus faecium DPC1146." Appl Environ Microbiol 65(4): 1506-1515.
8. Xiao, H., et al. (2004). "Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50." Microbiology 150(Pt 1): 103-108.
