Proof-of-Concept
1 The sensitivity analysis of bacterial strain
In order to ensure that the engineered bacterial strain can be obtained from antibiotic-resistant screening, the sensitivity of several types of antibiotics with EcN was tested. Under the same conditions, EcNs were inoculated into test tubes that respectively contained Chloramphenicol (30 μg/ mL), Ampicillin (50 μg/ mL), and Kanamycin (50 μg/ mL).
After 12 hours of incubation, the antibiotic that exterminated the wild-type EcN was chosen to be used in cloining screening. EcN was conlcuded to be most sensitive with Ampicillin.
2 Construction of expression vector
The recombinant plasmid SB1A3-MRFP from previous iGEM teams was modified to obtain high-copy component plasmids.
Under the effect of a special primer, the RFP gene sequence was successfully removed from the plasmid DNA sequence, and the NdeI, BamHI, and HindIII restriction sites were introduced. In this study, SB1A3-MRFP was first linearized by a BKL kit (Takala, Japan) to remove the RFP sequence and was then cycled under the Blunting Kination Enzyme named pSB18A. Through such a process, the recombinant plasmid pSB18A/INP-N-L was successfully transformed into the expressing strain, EcN-IL
3 Microscopic imaging analysis of expression vectors
As figure 3 shows, in order to know whether Lacc6 was successfully attached to the membrane of the bacterial strain, overlap PCR was used to connect the gene and the end of INP-N-Lacc6. Through the means of fluorescence microscopy, the recombinant strain ECN-ILG was observed to emit green fluorescence, while the control strain ECN-PSB18A remained dull. Thus, we concluded that Lacc6 anchored the surface of EcN cells.
4 The determination of enzyme activity and optimum reaction conditions of Engineering Strains
In order to evaluate laccase activeness in EcN-IL, the study used ABTS as a substrate to determine Lacc6 expression in whole-cell biocatalysts. Under an absorbance value of 420nm (Fig. 4), EcN-IL's Lacc6 expression was recorded to be 1.99 ± 087 U/ cell dry weight, while enzymatic activity was not detected in ECN - p18a and wild-type EN. The results showed that the laccase gene was successfully expressed on the strain's extracellular membrane and showed comparably high enzyme activity. Three replicates were set in the experiment.
Furthermore, this experiment investigated the effects of pH and temperature on the degradation efficiency of sulfadiazine of EcN-IL. As shown in Figure 4, the SDZ degradation rate rose as pH levels increased from 4.0 to 7.0. The degradation rate is maximized when pH is 7.0. Further increases in pH from this point result in a decrease in enzyme activity. Fig. 4 also shows that the degradation rate of SDZ peaks at 40 ℃. Therefore, the optimum temperature and pH conditions for Lacc6 are 40 ℃ and a pH of 7, or neutral.
Fig 3-3a and Fig show the degradation of sulfadiazine by different strains at multiple SDZ concentrations (30, 50, and 100 mg / L). EcN-Lacc6 and EcN-IL are both experimental groups that respectively perform intracellular and extracellular expression of Lacc6. EcN and PBS are both control groups, where one has the wild-type EcN and the other only with the solution. Antibiotic concentrations were taken after 3 hours through HPLC.
From 3-3 a, b, and c, it can be observed that Lacc6 expression did, in various degrees, reduce SDZ residual concentration. Between EcN-Lacc6 and EcN-IL, there were significant differences under 30mg/L and 100mg/L. The similarity of recorded residual at 50mg/L could be due to the probability that EcN-Lacc6 survived the endocytosis of SDZ long enough so that Lacc6 was able to catalyze degradation reactions. Despite there being a confounder, we were still able to conclude that the greatest amounts of SDZ were degraded when Lacc6 was expressed through the cell surface display technique.
Figure 3-4 displays sets of data gathered in the same experiment about the degradation rates of SDZ at the same incremental concentrations. The same four groups were tested for results. At 30mg/L, the greatest difference in SDZ degradation rate between EcN-IL and EcN-Lacc6 was observed. As concentrations increased, the differences between the two shortened. At 50mg/L, results were not significant, and at 100mg/L, results were barely significant.
A possible explanation for these occurrences (results at 50mg/L and 100mg/L) could be that the maximum degradation capacity of both engineered strains was similar due to the amount of Lacc6 expressed. Concentrations of SDZ at 50mg/L or more could have exceeded the maximum enzyme activity of Lacc6, thus producing similar results. Solely judging from the degradation rates at 30mg/L, we were able to conclude that EcN-IL had a greater degradation rate than EcN-Lacc6.
