Team:NTHU Taiwan/Proof
Result
E.coli Tolerance in PFOAWe cultured E.coli in liquid medium (3 ml LB and 5 ml E.coli broth) containing PFOA with different concentration and measured the growth curves via OD600. By mixing PFOA with 99% ethanol and series dilution, we get PFOA solution of different concentration.
The following is the results：
From the growth curves, we found that PFOA has little influence on the growth of E.coli.
Protein Expression Test of Fac-dexIn our experiment, after we transformed our plasmid into E.coli, we induced the expression of Fac-dex gene. If the gene is able to be induced, there should be a large quantity of Fac-dex existed in E.coli.
We used the induced E.coli culture as our samples. With the technic of SDS-PAGE, we can test if our plasmid can be expressed properly in E.coli or not.
We separately induced 8 different E.coli culture that had be transformed the Fac-dex gene. The molecular weight Fac-dex is about 34 kDa. As shown in fig 1, there are extra bands just below the 35 kDa marker in lane 4, 5, 6, 7. We use same E.coli which hadn’t been induced as the negative control in lane 1.
In conclusion, there are extra protein being expressed in the E.coli culture of lane 4, 5, 6, 7. The molecular weight of this extra protein is about 35 kDa. There are no expression of this protein in the negative control. This protein is most likely Fac-dex, and our plasmid is been properly induced.
Trace the DegradationPurpose: Test Fac-dex efficiency by tracing the degradation rate of PFOA
Principle:
Our core enzyme: Fac-dex
Fac-dex, under the category of Fluoroacetate Dehalogenase, has the ability to cause C-F bond cleavage. After Fac-dex breaking C-F bond, fluoride will be released, leading to the increase of fluoride concentration in water
The prediction of reaction mechanism
Therefore, by detecting the fluoride concentration with Fluorine Ion Selective Electrode in time, we can know the extent of PFOA degradation by Fac-dex. We induced E.coli culture that had be transformed the Fac-dex gene. Then the induced E.coli was transferred into new tube containing LB with different concentration of PFOA (As below). We also added Mg+2 as the cofactor of Fac-dex.
From the result above, the F- released by PFOA degradation is not detectable for PFOA concentration below 100(ppm), 0~12 hours. Based on fluoride-concentration-time graph from sample of modified E.coli (BL21 with Fac-dex) and PFOA of 500 ppm initially, we can tell that the concentration of fluoride increases in time due to the performance of PFOA degradation. It manifests that Fac-dex can successfully degrade PFOA and release fluoride.
E.coli tolerance in fluoride testWe inoculate starter culture at a 1:100 dilution into LB containing NaF with different concentration and measured the growth curves via OD600. We dissolved NaF 0.21g into 50ml and serial diluted the solution as follow 0.02M, 0.04M, 0.06M, 0.08M, 0.1M. The result is as below：
From the growth curve, we know that under 20mM of sodium fluoride, E.coli is able to grow normally. Under 60mM of sodium fluoride, the growth curve shifted slightly. Up to 80mM, the growth of E.coli is completely inhibited.
The function of Fluoride transporterFirst, we cultured the BL21 overnight, and then added IPTG to induce Crcb, Bpe, Fluc expression.
Second, we added different concentration of sodium fluoride (NaF) and used ELISA to detect the value of OD 600.
We think that if Fluoride transporter work, the bacteria will grow better.
Result: Compared with no plasmid control, we found a significant-different with 0.08M and 0.1M [NaF]. BL21 with Fluoride transporter, grew better than control.
Conclusion: First, the four figures show that if the environmental fluoride concentration increases, the bacteria which express fluoride transporter can growth better. Second, compare the three fluoride transporter family. We can find that Crcb family and Fluc is more efficiency to transport the fluoride out of the cell. So, we will use Crcb and Fluc to make our bacteria able to tolerate fluoride.
E.coli tolerance in wastewaterWe had the wastewater from the origin of factory. We grabbed one liter of it back to laboratory to test our engineered bacteria. We use fluoride electrode to test if concentration of fluoride rise. Our results didn't fit our expectation. Maybe there are too many unpredicted chemical compound in wastewater. So we can't test the fluoride concentrate solely and efficiently. Our maybe because the amount of our engineered bacteria added in wastewater isn't many enough to work in such amount of wastewater.
