Team:NPU-CHINA/Engineering
ENGINEERING
This year, NPU-China solve more than one problem in our project, and demonstrated our project using synthetic biology methods, and produced some expected results. Our project includes two systems: aflatoxin degradation system and Bacillus subtilis self-lysis system. Our team was reorganized this year and our unfamiliarity with experimental operations caused us to take a lot of detours, but in the end some satisfactory results were obtained.
-
Design and Build
Escherichia coli is a very mature expression system, can be used for the expression of a variety of proteins, and production of Escherichia coli medium is simple, Escherichia coli culture is easy, these advantages in practical application has a great advantage, so we prepare to choose Escherichia coli as the expression host of the Aflatoxin degradation enzyme BADE. Meanwhile, pET-28a (+) plasmid was selected as the vector, because the plasmid has lactose operon and T7 promoter, which can improve the expression of target protein. In addition, there are six histidine gene sequences before and after the cloning site, which can add His-tag to the amino terminal and carboxyl terminal of our target protein, providing conditions for subsequent separation and purification.
-
Test
After the construction of the vector plasmid, we transferred it into Escherichia coli BL21(DE3) competent cells for protein expression and enzyme activity measurement.
1.2.1SDS-PAGE electrophoresis during the purification of protein expression
Result: The expression of the target protein was improved after induction with IPTG, and the protein was successfully purified as the gragh shown below.
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.
1.2.2SDS-PAGE electrophoresis results of purified protein
Result: 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.
Figure 2: electrophoresis results
M:Protein marker; 1:8μg target protein; 2:6μg target protein1.2.3BCA method to determine protein concentration
Result: According to the BCA standard curve, the purified protein concentration was 3630 μg/mL. It can also be seen that a total of 21.78 mg of target protein can be purified in 400 mL of medium.
Figure 3: BCA standard curve
1.2.4ELISA kit for determination of specific enzyme activity
Result: The content of aflatoxin was 0.62 μg/L after 72 h by ELISA kit, and it was 1.99 μg/L in the blank group. About 66.8% of aflatoxin was degraded within 72 hours. It can be calculated that the specific activity is 0.2918 ng·h-1·mg-1 (the unit represents the amount of aflatoxin that can be degraded by 1 mg protein per hour).
Figure 4: Standard curve of relation between aflatoxin content and OD value
-
Learn
In the analysis of the above results, we found that BADE had a low degradation activity, and the enzyme could be modified to improve its degradation activity. Meanwhile, we learned that the induction effect of IPTG was greatly related to the amount of addition and induction temperature, and orthogonal experiments could be designed to obtain the optimal fermentation conditions and improve the expression level .Similarly, IPTG has certain toxicity to cells, which will be retained when induced expression is added into the bacterial solution. It is difficult to remove it, which will cause certain toxicity to the eating animals after adding into the feed. Therefore, direct induction of lactose can be considered in subsequent experiments.
-
Redesign
1.4.1Strategies for improving protein activity
①Rational design: By analyzing the active center of BADE, the key amino acids were determined. After BADE directional transformation by fixed-site mutation, the mutant gene sequence was cloned into vector plasmid pET-28a (+), and then transferred into Escherichia coli BL21(DE3) competent cells for protein expression purification and enzyme activity determination.
②Directed evolution: The mutant library was established by error-prone PCR, and the gene fragments in the library were cloned into the vector plasmid pET-28a (+). Then a high-throughput screening method was established to screen out the significantly improved enzyme activity, and finally transferred into E. coli BL21(DE3) competent cells for protein expression purification and enzyme activity determination.1.4.2Experimental method for determining the optimum conditions for IPTG induction
The purified protein was expressed under different conditions by orthogonal experiments, and the expression of the purified protein was compared by SDS-PAGE electrophoresis, so as to determine the optimal induction conditions.1.4.3Lactose induced protein expression assay
When lactose is added to LB liquid medium, lactose will be used as the only carbon source to induce protein expression. It is necessary to determine the amount of lactose and other carbon sources in the medium, so that the bacteria can precisely express protein at the end of logarithmic growth. Therefore, orthogonal experiments are also needed to determine.
-
Design
In order to simultaneously realize the expression of the degradation enzyme and the initiation of autolysis, we designed the following two protocols
2.1.1Conjured the aflatoxin degradation enzyme BADE to the P43 promoter. Because P43, as an integrated promoter in Bacillus subtilis, does not need to be added as inducer compared with T7 and lactose operon, so it will be safer when adding feed.
The lytic protein (BsrG, lytC) and the lytic protein promoter (PyqfD, PmmgA) were conjugated to each other by pin-pair combination.
Finally, the degradation enzyme part and the lyase part were simultaneously cloned into the pHT01 plasmid, as shown in the figure. It can simultaneously express aflatoxin degradation enzyme, and express lysate protein after a certain period of time, start autolysate to release aflatoxin degradation enzyme, and finally complete the degradation of aflatoxin in the feed.
2.1.2According to the investigation, the pAX01 plasmid can be integrated into the genome of Bacillus subtilis, so four combinations of 1 lysin and lysin promoter can be cloned into the pAX01 plasmid respectively, and then integrated into the genome after transfer into Bacillus subtilis. The BADE gene of aflatoxin degradation enzyme was cloned into pHT01 by connecting with the P43 promoter. Finally, the constructed plasmid was transferred into Bacillus subtilis integrating with lytic protein, as shown in the figure. The expression and autolysis of degradation enzymes were realized.
-
Bulid
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.
-
Test
2.3.1Growth curve
We added WB600 to LB medium for culture and measured its growth curve. 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 there was no obvious curve decline in the experimental group during the stable period (the growth curve is shown in FIG 5).
Figure 5: Growth curve 1 shows the control group transferred into empty plasmid. 2-4 were transferred to the experimental group with two target genes
2.3.2SDS-PAGE
Meanwhile, after 24 h, the bacterial solution was prepared into the bacterial fragmentation solution for SDS-PAGE electrophoresis. The results showed that there was no significant difference in protein expression level, indicating that the expression levels of degradation enzyme and lysate protein were not significantly increased (as shown in Figure 6).
Figure 6: Protein SDS-PAGE electrophoresis
M: protein marker; 1: The cell fragmentation supernatant in 1 mL of the control group after 24h culture; In experimental group 1, the supernatant of bacterial fragmentation was obtained in 1 mL bacterial solution after 24h culture. In experimental group 2, the supernatant of cell fragmentation was obtained in 1 mL bacterial solution after 24h culture. In experimental group 3, the supernatant of thallus fragmentation was obtained in 1 mL bacterial solution after 24h culture
2.3.3Enriching Selenium
Sodium selenite was added to the medium, and then the constructed expression host was inoculated for cultivation. After 24 h, the expression host was centrifuged to obtain Figure 7. It can be seen that the expression host we constructed still has the effect of enriching selenium.
Figure 7: The centrifuge tube on the left is the uncentrifuged bacterial solution;
the centrifuge tube on the right is filled with the centrifuged bacterial solution -
Learn
After discussing the experimental results with our instructor, we found that it may be the ribosome binding site followed by the connection of the target gene, resulting in the ribosome unable to correctly bind and perform functions. This resulted in no significant difference in the expression levels of degraded and lysed proteins.
-
Redesign
2.5.1Redesign the number of bases between ribosome binding sites and genes. To ensure that ribosomes bind properly and perform translation functions.
2.5.2Determine what site the lytic protein gene is connected to on pAX01, clone the target gene into pAX01 by homologous recombination method, and then transfer it into WB600.
2.5.3Determine the growth curve of the expression host obtained by the two methods, compare the differences between the two methods, and select the better method
2.5.4The combination of the remaining three lysins and lysin promoters was selected according to method 3 to construct expression host. The growth curve was also measured. Select the best self-cracking combination.
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
