Team:Think Edu China/Proof Of Concept

The sensitivity analysis of bacterial strain

In order to ensure the engineered bacterial strain can be obtain by the antibiotic resistant screening, the sensitivity several types of antibiotics Under the same conditions, inoculatEN into test tubes that contains Chloramphenicol (30 μg/ mL), Ampicillin (50 μg/ mL) and Kanamycin (50 μg/ mL).

After 12 hours of incubation, if the wild ECNS cannot survive in the presence of the above antibiotics that is used for cloning screening ( Figure 2.1), the strain can then be used

Fig 21 The sensitivity of EcN to several antibiotics；Amp+, Kna+, Cn+（-）.

Construction of expression vector

As shown in the figure, the recombinant plasmid SB1A3-MRFP from IGEM was modified in order to obtain high copy component plasmids.

Under the effect of 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 linearized by BKL kit (Takala, Japan) to remove the RFP sequence, and then the plasmid was cycled under the of Blunting Kination Enzyme named pSB18A. Finally, the recombinant plasmid pSB18A/INP-N-L was successfully transformed into the expressing strain EcNECN-IL

Fig 2-2 The map of recombinant plasmid

Microscopic imaging analysis of expression vectors

As figure 23 shows,in order to whether gene was successfully anchored on the membrane of the bacteria strain, overlap PCR to connect gene and the end of INP-N-Lacc6. fluorescence microscopy, the recombinant strain ECN-ILG observed to emit green fluorescence, while the control strain ECN-PSB18A . Thus, came to the conclusion that Lacc6 anchored the surface of EcN cells.

Fig 23 Fluorescence microscopic imaging of engineering strain. a,b: the reporter plasmid expressed green fluorescent protein; c,d: bright field and dark field imaging of the control strain

3.3.1 determination of enzyme activity and optimum reaction conditions of Engineering Strains

In order to evaluate the laccase activit in EN, the study used ABTS as substrate and determined the laccase activity of whole cell catalyst was 1.99 ± 087 U/ cell dry weight when the absorbance value was 420nm (Fig. 3-1). , the enzyme activity was not detected in ECN - p18a and wild-type EN. The results showed that laccase gene was successfully expressed on extracellular membrane and showed high enzyme activity. Three replicates were set in the experiment.

Fig 3-1. The Laccase enzyme activity of the whole-cell biocatalyst.

Furthermore, this experiment investigated the effects of pH and temperature on the degradation of sulfadiazine by engineering strains. in Figure 3-2the pH is in the range of 47, the degradation rate of ECN on SDZ gradually increases, and reaches the maximum when pH 7.0. With the gradual increase of pH, the degradation efficiency decreases Fig. 3-2 also shows that the degradation rate of SDZ increases gradually with the increase of temperature, and highest 40 ℃. Therefore, the optimum temperature 40 ℃.

Fig 3-2 Effects of different pH and temperature on the degradation of sulfadiazine.

Ability of engineering strain to degrade sulfadiazine

Fig3-2a, andshow the degradation of sulfadiazine by whole cell biocatalyst at different concentrations (30, 50 and 100 mg / L). After 3 hours, the antibiotics was detected by HPLC. Seen from the figure the sulfadiazine in c and acc6 group w less than that in the control group. The results showed that the whole cell catalyst could degrade sulfadiazine.

Comparing the sulfadiazine in c- and -acc6 groups, it was found that there was no difference. Therefore, proved the existence of protein had no effect on the expression of acc6 proteiFigure 3-3a, and show the analysis of the degradation at different concentrations (30, 50 and 100 mgL) by . the control EN group, the degradation ratecan reach 30 ± 2.3%.