Team:NPU-CHINA/Result
RESULT
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SDS-PAGE electrophoresis
① SDS-PAGE electrophoresis during the purification of protein expression
Figure 1: Electrophoretic results
M: Protein marker; 1: Thallus crushing solution before IPTG induction; 2: Thallus crushing solution after IPTG induction; 3: Supernatant after ultrasonic fragmentation of thallus; 4: Precipitation after ultrasonic crushing of thallus; 5: The outflow of supernatant through the column; 6: The last eluent; 7: Target protein.
Result 1: According to the electrophoretic diagram, the expression of the target protein was improved after induction with IPTG, and the protein was successfully purified.
② SDS-PAGE electrophoresis results of purified protein
M:Protein marker; 1:8μg target protein; 2:6μg target protein
Result 2: According to the electrophoretic diagram, the molecular weight of the purified protein was between 35-48 kD, which was close to the theoretical value, indicating that the protein was successfully purified.
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Protein concentration determination
Figure 3: BCA standard curve
Results 3: The concentration of purified protein was 3630 ug/mL by BCA method. It was also known that 21.78 mg of target protein was purified in 400mL medium.
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Used ELISA kit to determine the specific enzyme activity
Figure 4: Standard curve of relation between aflatoxin content and OD value
Result 4: Aflatoxin content was 0.62 ug/L by ELISA kit after 72 h, and 1.99 ug/L in blank group. About 66.8% of aflatoxin was degraded within 72 h. The specific activity was calculated to be 0.2918 ng·h-1·mg-1 (unit is the amount of aflatoxin that can be degraded by 1 mg protein per hour)
Until now, we complete BADE expression of aflatoxins degradation enzyme purification and the determination of enzyme activity.It can be seen that BADE for aflatoxins degradation effect is good, can be on the low concentration of aflatoxin substrate degradation effectively. Thus, it can decrease the concentration of aflatoxin into a safety level which is very important for the actual feed. Because the content of aflatoxin in the actual feed is very easy to be exceeded by the environment and then cause a lot of losses, but BADE can completely reduce the final content to a safe range. At the same time, we noticed that the degradation time slightly longer.Therefore, in the final use, we need to add the enzyme into the feed in advance to ensure that the BADE has sufficient time for the degradation of aflatoxin.
In the future, research will focus on the enzyme activity of promotion. We can adopt the method of directed evolution of enzyme is modified. Such as: build mutant library by error-prone PCR, gene cloning from the library to the carrier in the plasmid, then a high-throughput screening method was established to extract enzyme activity was significantly improve, finally to determine the expression and purification and enzyme activity. So in the use of aflatoxins degradation enzyme will be more time saving, have higher economic effect.
What we did:
Verified that Bacillus subtilis can indeed metabolize sodium selenite to produce nano-selenium
Evaluated the effects of different concentrations of sodium selenite on the normal growth of
Bacillus subtilis.
Determined the optimum concentration of sodium selenite for selenium production.
Verified that the selenium produced is nanoscale.
Determined the selenium production efficiency of Bacillus subtilis.
Not completed: Use XRD and SEM to analyze whether the selenium is nanoscale.
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Growth curve drawing of Bacillus subtilis
The concentration of bacteria is roughly proportional to its absorbance at 600nm.In order to determine the logarithmic growth period of the selected engineered bacteria, we measured the OD600nm of WB600 at different growth times (0h, 2h, 4h...24h) , and the growth curve of WB600 was plotted.(Fig.5)
Figure 1: Electrophoretic results
Figure 5. Growth curve of Bacillus subtilis WB600
It can be seen that OD600nm rises sharply at 4-8h. According to the characteristics of the logarithmic growth period of bacteria, Bacillus subtilis reach the logarithmic growth period during 4-8h culture. -
Intracellular transformation of sodium selenite
We added sodium selenite into LB liquid medium and set up the final concentration gradient of sodium selenite (0mM、0.6mM、0.8mM、1.0mM、1.2mM).Finally, the fermentation liquid with sodium selenite was red, and the fermentation liquid without sodium selenite was yellow (Fig.6 and 7),indicating that elemental selenium was generated in the fermentation broth.The bacteria were separated from the culture medium by centrifugation, and it was found that only the precipitate appeared red, indicating that the elemental selenium was produced in the bacteria and remained inside or on the surface of the bacteria.
Figure 6.Uncentrifuged gradient concentration of sodium selenite fermentation broth
Left 1: fermentation broth without sodium selenite;Left 2: fermentation liquid with the final concentration of sodium selenite of 0.6mM;Left 3: fermentation liquid with the final concentration of sodium selenite 0.8mm;Left 4: fermentation broth with the final concentration of sodium selenite of 1.0mm;Left 5: fermentation liquid with the final concentration of sodium selenite of 1.2mm;
Figure 7. Centrifuged gradient concentration of sodium selenite fermentation broth
Left 1: fermentation broth without sodium selenite added after centrifugation;Left 2: 0.6mM sodium selenite fermentation broth after centrifugation;Left 3: 0.8mm sodium selenite fermentation broth after centrifugation;Left 4: 1.0mm sodium selenite fermentation broth after centrifugation;Left 5 :1.2mM sodium selenite fermentation broth after centrifugation;
In order to evaluate the effect of sodium selenite concentration on cell growth, we carried out solid medium co-culture experiment, co-culture growth curve experiment and domestication experiment respectively.
In the solid medium co-culture experiment, we added sodium selenite to LB solid medium, set the final concentration of sodium selenite (0.5mM, 1.0mM, 1.5mM, 2.0mM), WB600 grown to plateau stage is coated with 10 times, 100 times and 1000 times dilution ratio.Single colonies were counted after 24h culture(Fig. 8).The final results showed that the 1.0mm group had the largest number of colonies.
In the co-culture growth curve experiment, sodium selenite was added into LB liquid medium, set up the final concentration gradient of sodium selenite(0mM、0.6mM、0.8mM、1.0mM、1.2mM).By measuring the OD600nm of WB600 at different growth time(0h、2h、4h……24h),the growth curves of WB600 in different concentrations of sodium selenite were plotted(Fig.9).It can be seen from the growth curve that the higher the concentration of sodium selenite, the later WB600 reached the plateau, but the final OD600nm didn't differ much.
In the domestication experiment, WB600 was streaked in solid medium with high concentration of sodium selenite,after 24h culture, the colonies were cultured in solid medium with higher concentration of sodium selenite, and increasing the concentration,it was found that bacterial colonies could still grow at 50mM sodium selenite concentration(Fig. 10).In summary, we believe that WB600 has a strong tolerance to sodium selenite, and the low concentration of sodium selenite has a limited effect on the growth of WB600.
Figure 8. Culture of Bacillus subtilis in sodium selenite solid medium with different concentration gradient
4A: 0.5mM sodium selenite solid medium for cultivation of Bacillus subtilis at different dilution rates;
4B: 1.0mM sodium selenite solid medium with different dilution ratio;
4C: 1.5mm sodium selenite solid medium with different dilution ratio.
4D: 2.0mM sodium selenite solid medium with different dilution ratio.
Figure 9. Growth curves of WB600 in different concentrations of sodium selenite
Figure 10. Domestication experiment
Upper left: the growth of Bacillus subtilis in sodium selenite solid medium of 15mM;Top right: 20mM sodium selenite solid medium Bacillus subtilis growth;Lower left: 30mM sodium selenite solid medium growth of Bacillus subtilis;Lower right: The growth of Bacillus subtilis in sodium selenite solid medium of 50mM; -
Characterization of selenium
Fermentation broth with different concentrations of sodium selenite cultured for 24h was removed from (2) and transferred 2 ml in test tube,digested with concentrated nitric acid at 100℃ for 48h, then filtrated with 0.22μm bacterial membrane, prepare atomic absorption spectrum samples.Fermentation broth with different concentrations of sodium selenite cultured for 24h was removed from (2) and transferred 1.5 ml in 1.5mL centrifuge tubes,centrifuge 12500g for 10min, discard supernatant, add electron microscope fixative solution to full, prepare TEM sample.The two groups of samples were sent to Scientific Compass inspection Organization for testing.
Calculate the yield:Yield=The measured nano-selenium mass / Selenium content of sodium selenite*100%
The sodium selenite concentration(c0)-product selenium concentration (cx)- conversion rate (η) curve was plotted according to the data.The results showed that the selenium concentration increased with the increase of sodium selenite concentration, and the increase rate slowed down when the concentration of sodium selenite reached 1.0mM.The conversion rate peaked at 0.6mm and gradually decreased (Fig.11).
Figure 11. Production concentration and conversion of selenium in liquid culture medium with different concentrations of sodium selenite
The system consists of two parts -- the promoter PyqfD and the lysis protein BsrG.When Bacillus Subtilis is in the growth phase, the lytic protein BsrG is not expressed and the bacteria will not die.When Bacillus subtilis grows to stationary phase, the promoter PyqfD begins to express, thus initiating the expression of the lysis protein BsrG, which leads to bacterial lysis due to the toxicity of BsrG protein.
What we did:
Verified that the transcription of PyqfD was initiated only during the bacterial stationary
phase.
Verified that BsrG has the function of killing Bacillus subtilis.
Determined the killing effect of BsrG on Bacillus subtilis.
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System construction
We first extracted the whole genome from Bacillus subtilis WB600, designed primers for gene fragments according to the method recommended by Gibson assembly method, and then obtained the DNA fragments of PyqfD and BsrG required by the system through PCR.A small amount of PCR products were extracted and agarose gel electrophoresis was used to verify that the WB600 genome contained the gene fragment needed for the experiment(Fig.12).The results of agarose gel electrophoresis showed that the fragment required for the lysis system we constructed existed in the WB600 genome.
Figure.12 Verify the presence of target genes in WB600 genome by agarose gel electrophoresis
Designed the reverse primers of the plasmids according to the method recommended by Gibson assembly method, and linearized plasmids were obtained by PCR technology from the pHT01 plasmids constructed by the company.After that, 1μ L DpnⅠenzyme or DMT enzyme was added into the PCR reaction system, and the reaction temperature was 37°C for 1h to digest the circular plasmids in the product, so as to reduce the probability of false positive in subsequent experiments.
The PCR products were purified by agarose gel electrophoresis (Fig.13),and DNA was purified by gel recovery kit.By homologous recombinant cloning, linearized plasmids and gene fragments were connected to form circular plasmids inserted into foreign genes for bacterial transformation.
Figure.13 Purify DNA by agarose gel electrophoresis
The homologous recombinant product was directly transformed into E.coli-DH5α competent cells at the volume of 1/10.Amp - resistant solid medium was used to screen positive recombinants.Amp resistant solid medium was used to screen positive recombinants (Fig. 14), and then the single colony was transferred to liquid medium for culture. After 1h culture, a small amount of bacterial liquid was extracted and the inserted fragment was amplified by colony PCR technology, the colony PCR products were verified by agarose gel electrophoresis(Fig.15).If false positive recombination, electrophoresis results showed no bands;If the recombination is true positive, electrophoresis results show that there are obvious bright bands in the corresponding gene size.The experimental results showed that the lysis system was successfully constructed, and it was not because of false positive that the AMP-resistant solid medium formed a single colony.
Figure.14 DH5α positive recombinants on AMP - resistant solid medium
Figure.15 Detected colony PCR results by agarose gel electrophoresis
True positive recombinants were obtained for extended culture,after 16 hours culture, plasmid pHT01 was extracted from the bacterial solution, and then transferred into the engineering bacterium WB600, which was cultured on solid medium with chloramphenicol resistance and screened for positive recombinants(Fig.16).In order to verify whether it was a true positive recombinant, single colonies were selected from solid medium containing chloramphenicol and transferred to liquid medium for culture.About 1 hour later, a small amount of bacterial solution was taken to amplify the inserted fragment by colony PCR technology, and then agarose gel electrophoresis was used to observe whether there were bright bands in the positions with equal gene size.The results showed that,in the WB600 transformation experiment, we successfully transformed the constructed plasmid into WB600 host bacteria.
Figure.16 WB600 positive recombinants on solid culture medium for chloramphenicol resistance
We conjured P43 to BADE and PyqfD to lytC by fusion PCR, and cloned both of them into pHT01 plasmid by homologous recombination. The plasmid was then transferred into the prepared WB600 receptor cells.
1、However, when the growth curve of the prepared expression host was measured, it was found that the growth curve of the experimental group was almost the same as that of the control group transferred into empty plasmid, and the growth curve of the experimental group did not decrease significantly in the stable period (as shown in FIG. 17).
Figure. 17 Growth curve 1 shows the control group transferred into empty plasmid. 2-4 were transferred to the experimental group with two target genes
2、Meanwhile, the results of SDS-PAGE showed no significant difference in protein expression, indicating that the expression levels of degradation enzyme and lysate protein were not significantly increased (as shown in FIG. 18).
Figure.18 SDS-PAGE electrophoresis of protein M: protein marker; 1: The cell fragmentation supernatant in 1 mL of the control group after 24h culture; Experimental group 1: the supernatant of bacterial fragmentation was obtained in 1 mL bacterial solution after 24h culture. Experimental group 2: the supernatant of cell fragmentation was obtained in 1 mL bacterial solution after 24h culture. Experimental group 3: the supernatant of thallus fragmentation was obtained in 1 mL bacterial solution after 24h culture
3、Sodium selenite was added to the medium, and then the constructed expression host was inoculated into the medium for culture. After centrifugation for 24 h, figure 3 was obtained, indicating that the expression host constructed by us had selenium enrichment effect.
FIG. 19 Left measuring centrifuge tube is the bacterial liquid without centrifugation; The centrifuge tube on the right is filled with centrifuged bacterial liquid
CONTACT NPU CHINA
NPU-CHINA is the iGEM team of Northwestern Polytechnical University
Address: 127 West Youyi Road Beilin District, Xi'an,
Shaanxi 710072, China
Contact: npuigem@outlook.com
bilibili: NPU-iGEM
