Difference between revisions of "Part:BBa K2382002"

FGD ( plasmid is from Australia) were transformed into E. coli BL21 (DE3) strain to express the protein. Then IPTG was used to induce the expression system, since the plasmid in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we do western blot. The results are demonstrated in figure 1. After centrifuging for two times, we could find a high percentage of proteins in the pellet (the 9500 P group) and small amount of protein exist in the supernatant ( the 13000T group ). FGD ( plasmid is from Australia) was transformed into E. coli BL21 (DE3) strain to express our protein. Then IPTG was used to induce the expression system since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we did SDS-PAGE electrophoresis and coomassie brilliant blue staining. The results are demonstrated in the Fig. 1C. After centrifuging two times, we could find a high percentage of proteins in the cell supernatant (the 13000 Su group).

Fig 1C:Cell lysates were analyzed by SDS-PAGE and coomassie brilliant blue staining. 9500 T meant the initial sample obtained after sonication; 9500 P and 13000 T meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant the pellet and the supernatant obtained after 13000 rpm for 20 min.(C) Samples contained Australian and synthetic FGD.

Protein purification, and dialysis

After extracting the cell lysates, we used nickel-resin column to purify our target proteins from the cell lysates because all of our proteins were tagged with 6 histidines at their C-terminal ends. After protein purification, protein dialysis with diaysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH=7.5), and 20% glycerol to remove imidazole in our purified proteins, we did SDS-PAGE gel electrophoresis to ensure our target proteins were successfully purified (Fig. 2A ). The molecular weights of these proteins are listed in the Table 1. The standard BSA proteins were used to quantify the concentration of target proteins.

Fig. 2A: Concentration of proteins was quantified by SDS-PAGE and standard BSA samples with 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 mg/ml. Two recombinant proteins were expressed by the plasmids from Australia.

Protein solubility analysis

To know whether the solubility of our two enzymes (MSMEG_5998 and FGD BBa_K2382002) increased after fusing enzymes with thioredoxin, we dissolved all cell lysates which containing pellet and supernatant and did western blot to detect the content of our target proteins. All proteins were detected by anti-6x His Tag antibody because all of them contained a 6-histidines tail when bacteria expressed them. In Fig. 3, we could find there was good expression of both Australian and synthetic MSMEG5998 in the “13000 Su” group when compared with the “13000 P” group. This result meant that most proteins were dissolved in the supernatant while few proteins deposited in the cell pellet after 13000-rpm centrifugation. However, we could not observe good solubility in both Australian and synthetic FGD because there were little or no difference between the “13000 Su” group and the “13000 P” group.

Figure 3: Cell lysates in the process of two times centrifuge were analyzed by western blot. The abbreviations of five groups were the same as Fig. 1.

Enzyme Function Results

Enzyme Activity Assay

The conditions of reaction to degrade aflatoxin by MSMEG5998 were modified from Taylor’s study[6]. All concentrations of reactants are listed in Table 2 and 32 μM aflatoxin was used. We first mixed all reactants in eppendorfs and then put them at 22°C.

Figure 7A: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (A) Direct 365 nm absorbance were detected after mixing Australian/synthetic MSMEG5998 and Australian/synthetic FGD and other reactants at 0th and 8th hour in the environment of pH=7.5 and 22℃.

In Fig. 7A, we compared two proteins, MSMEG5998 and F420-dependent glucose-6-phosphate dehydrogenase (FGD) expressed from Taylor’s vectors (from Australia) and from our synthetic vectors. We found that both the Australian and synthetic MSMEG5998 have great activity and degraded aflatoxin B1 by more than 60%. The effect of the synthetic one may be better than the Australian one but there were no statistic significance.

However, only Australian FGD has activity to reduce F420 into F420H2 and help the reaction. This finding corresponds with our dry lab results. Therefore, we used Australian and synthetic MSMEG5998 and Australian FGD to do the same experiment again to figure out whether the degradation percentage was dependent of time and whether the main reason of degradation was MSMEG5998. </p>

Figure 7B: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (B) The same way as (A) but Australian/synthetic MSMEG5998 and Australian FGD were used and the reaction were detected at 0th, 2nd, 4th, 6th, and 8th hour. a, p < 0.001 compared to the 0th hour of the synthetic MSMEG5998(+) group; b, p < 0.001 compared to the 0th hour of the Australian MSMEG5998(+) group; c, p < 0.001 compared to the same time of the Australian MSMEG5998(+) group.

Figure 7C: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (A) Direct 365 nm absorbance were detected after mixing Australian/synthetic MSMEG5998 and Australian/synthetic FGD and other reactants at 0th and 8th hour in the environment of pH=7.5 and 22℃. (C) The same way as (B) but the degradation percentage were detected by ELISA. Because the initial concentration of aflatoxin (10000 ng/ml) was too high to be detected by the ELISA, we didn’t demonstrate the initial data.

The results were detected by direct 365 nm absorbance (Fig. 7B) and by ELISA (Fig. 7C). We found out that the degradation percentage was time-dependent. The synthetic MSMEG5998 had better activity than Australian MSMEG5998. The former was able to degrade 83% aflatoxin after 8 h while the latter could only degrade 52% aflatoxin.

References

(1)Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.

(2)Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.

(3)Bashiri G, Rehan AM, Greenwood DR, Dickson JMJ, Baker EN. Metabolic Engineering of Cofactor F420 Production in Mycobacterium