After producing our target protein---bacteriocin, we investigated the function of bacteriocins with 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 used Inhibition Zone to measure the effective inhibition of bacteriocins. We used the condensed culture spread to the agar plate, added bacteriocins and let it dry. In that case, the dose of inhibition zone was as same as microdilution which could 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, then centrifuged products which had been sonicated and collected supernatants as samples. The positive control in this experiment is Ampicillin and the negative control is Bacillus subtilis without adding bacteriocins. We used Elisa Reader to record O.D.600nm values of samples once an hour, if OD values decrease or keeps at the same value, it means that the bacteriocin inhibited the growth of Bacillus subtilis. Further, we can also know when does bacteriocin degraded by observing the growth curve of Bacillus subtilis. The pre-test data are all triplicated and normalized.
Inhibition Analysis of purified bacteriocins
Figure 1：Normalized growth curve of purified Leucocyciclin Q.
Figure 2：Normalized growth curve of purified Enterocin B.
Dose Response Assessment of
Purified Bacteriocin (Inhibition Zone)
Figure 3：Inhibition zone test of Enterocin B for four dosages.
A: Enterocin B 1.0
B: Enterocin B 0.5
C: Enterocin B 0.25
D: Enterocin B 0.125
E: Negative Control
Figure 4：Inhibition zone test of Leucocyclicin Q for four dosages.
A: Leucocyclicin Q 1.0
B: Leucocyclicin Q 0.5
C: Leucocyclicin Q 0.25
D: Leucocyclicin Q 0.125
E: Negative Control
Figure 5：Inhibition zone test of Enterocin 96 for four dosages.
A: Enterocin 96 1.0
B: Enterocin 96 0.5
C: Enterocin 96 0.25
D: Enterocin 96 0.125
E: Negative Control
Concentration of Enterocin B 1.0 is 263mg/L, Leucocyclicin Q 1.0 is 217mg/L and Enterocin 96 1.0 is 74mg/L. Figure3~5 show that Enterocin B, Leucocyclicin Q and Enterocin 96 have significantly inhibition range comparing to negative control, column buffer, which is the buffer that protein solubilize in after purification.
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 7 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 has is both effective and stable.
Durancin TW-49M
Figure 8：Bar diagram that showed percentage resistance of Durancin TW-49M to Bacillus subtilis after acting for four hours. Durancin TW-49M (18.94%)
Figure 9：Inhibition Zone Test of Durancin TW-49M. A: Durancin TW-49M; B: Negative Control
Figure 10：Normalized growth curve of Bacillus subtilis that showed Durancin TW-49M inhibiting ability throughout four hours.
Enterocin B
Figure 11：Bar diagram that showed percentage resistance of Enterocin B to Bacillus subtilis after acting for four hours.
Enterocin B(44.24%)
Figure 12：Inhibition Zone Test of Enterocin B
A: Enterocin B; B: Negative Control
Figure 13：Normalized growth curve of Bacillus subtilis that showed Enterocin B inhibiting ability throughout four hours.
Lacticin Z
Figure 14：Bar diagram that showed percentage resistance of Lacticin Z to Bacillus subtilis after acting for four hours.
Lacticin Z (96.32%)
Figure 15：Inhibition Zone Test of Lacticin Z.
A: Lacticin Z
Figure 16：Normalized growth curve of Bacillus subtilis that showed Lacticin Z inhibiting ability throughout four hours.
Enterocin 96
Figure 17：Bar diagram that showed percentage resistance of Enterocin 96 to Bacillus subtilis after acting for four hours.
Enterocin 96 (45.16%)
Figure 18：Inhibition Zone Test of Enterocin 96
A: Enterocin 96; B: Negative Control
Figure 19：Normalized growth curve of Bacillus subtilis that showed Enterocin 96 inhibiting ability throughout four hours.
Bovicin HJ50
Figure 20：Bar diagram that showed percentage resistance of Bovicin HJ50 to Bacillus subtilis after four hours.
Bovicin HJ50 (82.05%)
Figure 21：Inhibition Zone Test of Bovicin HJ50.
A: Bovicin HJ50; B: Negative Control
Figure 22：Normalized growth curve of Bacillus subtilis that showed Bovicin HJ50 inhibiting ability throughout four hours.
Leucocyclicin Q
Figure 23：Bar diagram that showed percentage resistance of Leucocyclicin Q to Bacillus subtilis after four hours.
Leucocyclicin Q (40.58%)
Figure 24：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 25：Bar diagram that showed dose response of Enterocin 96 after four hours.
Figure 26：Bar diagram that showed dose response of Leucocyclicin Q after four hours.
Figure 27：Bar diagram that showed dose response of Enterocin B after four hours.
Figure 28：Bar diagram that showed dose response of Bovicin HJ50 after four hours.
Figure 29：Bar diagram that showed dose response of Durancin TW-49M after four hours.
Figure 30：Bar diagram that showed dose response of Lacticin Z after four hours..
Figure25~30 show dose response assessments of six bacteriocins. Durancin TW-49M and Enterocin B represent a greater dose response data. After that, we analyze the target band of SDS-PAGE gel and estimate concentration of bacteriocins.
Enterocin 96_1.0X：21.12mg/L, Durancin TW-49M_1.0X is 24.32mg/L, Enterocin B_1.0X is 21.12mg/L, Bovicin HJ50_1.0X is 18.71mg/L and Lacticin Z_1.0X is 260.29mg/L.
Figure 31：The relative percentage of O.D.600nm value of bacteriocins.
P values were used to determine whether two sets of data were significantly different with each other or not. Therefore, the smaller p-value was, the more effective that bacteriocin was to inhibit the growth of Bacillus subtilis than negative control does. The star indicates p< 0.05 and two stars indicate that p< 0.01.
Figure 31 showed the p-value and the star of each sample. P-value of Durancin TW-49M was the smallest in the figure, which indicated that it was the most reliable.
Conclusion
From our experiments we concluded that our 6 bacteriocins were functional. We began performing both functional test in 96-well plate and inhibition zones. Our results indicated successful inhibition of Bacillus subtilis. Then, we moved on to dose response assessment. Four of our six peptides showed convincing results, following the trend of greater inhibition with higher dosage.
