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 used supernatants 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 OD 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 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.
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 rangecomparing 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 1：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 2：Normalized growth curve of bacteriocins and Negative Control.
- Figure 1 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 2 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.
- The following data showed that each bacteriocin can have effective function without purifying, keeping costs of using bacteriocins as a biostimulator 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.
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 12：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 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 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 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.
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 are used to determine whether two sets of data are significantly different with each other or not. Therefore, the smaller p-value is, which means bacteriocin exactly more effective 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 shows the p-value and the star of each sample. P-value of Durancin TW-49M is the smallest in the figure, which indicates it is the most reliable.
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.
