Difference between revisions of "Team:NCTU Formosa/Wet Lab/Functional Analysis"

     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.

     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 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.

     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.

     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.

     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.

     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. Of our six peptides, four showed convincing results, following the trend of greater inhibition with higher dosage.