Team:NPU-CHINA/Design
DESIGN
Escherichia coli is a very mature expression system, which 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.
Finally, we inserted the BADE gene sequence between Nedl and Xhol cleavage sites of restriction enzyme on PET-28A (+) and retained the amino and carboxyl end His-tag.
In the process of protein expression and purification, IPTG was used to induce the expression, and affinity purification was carried out by nickel column. Finally, SDS-PAGE electrophoresis was used to visually express the expression and purification of the target protein, and BCA method was used to determine the protein concentration after purification .
For the determination of BADE specific activity of aflatoxin degradation enzyme, we plan to use ELISA kit to detect the residual aflatoxin content after a period of time of aflatoxin reaction between enzyme and substrate, so as to calculate its specific activity.
（Se）is an essential trace element for human body.Chinese Nutrition Society lists selenium as one of the 15 essential nutrients for human body. A large number of clinical experiments at home and abroad show that selenium deficiency in human body can cause dysfunction of some important organs and lead to many serious diseases.More than 40 countries in the world are in selenium deficiency areas, and hundreds of millions of people in 22 provinces of China are in selenium deficiency or low selenium zones, where the population has a high incidence of cancer, liver disease, cardiovascular disease and other diseases.Selenium is also indispensable in livestock, so we hope to make engineered bacteria produce nano-selenium that can be absorbed by livestock.
Since the gene expressing this function is the engineering bacterium's own, we don’t need to transform the exogenous gene into the engineering bacterium through gene recombination technology.We just add the toxic sodium selenite to the culture,Bacillus subtilis will undergo morphological changes under the stress of sodium selenite, and degrade the toxic sodium selenite into non-toxic nano-selenium particles.
Finally, XRD, SEM, TEM methods to confirm that the selenium produced is nanoscale selenium.
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Background
The engineered bacteria needed to secrete aflatoxin degrading enzymes（BADE）out of the cell，however, when Bacillus subtilis is used to overexpress some enzyme molecules and synthesize some intracellular metabolites,often these products will be not secreted in large quantities out of the cell for some reasons of their own nature.Due to the characteristics of Gram-positive bacteria, Bacillus subtilis has a thick peptidoglycan layer outside the cell, and the crushing process is time-consuming and laborious, and easy to cause the target product denaturation.In practice, engineered bacteria need to be eaten by livestock,thus we could not find a suitable condition from the extracellular to regulate the lysis of bacteria.Therefore, we looked for the trigger conditions within the bacteria to make the lysis pathway of Bacillus subtilis simple and efficient.Based on literature research, we designed a self-lysis expression host to help engineering bacteria better perform the function of degrading aflatoxin.
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Different combinations of lysis schemes
Before the experiment, we could not determine the transcription efficiency of different promoters and the lysis effect of different lysis protein.Therefore, we designed several combinations of different promoters and lysis proteins based on literature research(Table.1).lytC belongs to autolysin，which is reported to be involved in cell division, separating progenitor cells by degrading peptidoglycan that divides the septum, and overexpression can be used for Bacillus subtilis lysis.BsrG-SR4 is a toxin-antitoxin system(TA), BsrG is the toxin in this system. Overexpression of BsrG can also be used for Bacillus subtilis lysis.Through literature search, it was found that there were more reports on promoter PyqfD, so the transcriptional efficiency of promoter PyqfD was expected to be higher than that of promoter PmmgA, so promoter PyqfD was the first choice and promoter PmmgA was the alternative.
Promoter Lysis protein 1 PyqfD BsrG 2 PyqfD lytC 3 PmmgA BsrG 4 PmmgA lytC Table.1 Combinations of different promoters and lysis proteins
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Construction of autolysis system
The system consists of the PyqfD promoter from Bacillus subtilis WB600 and lysis protein,and was expressed on the pHT01 vector.The PyqfD promoter is regulated by a signal factor expressed in the stationary phase of bacteria.In the process of engineering bacteria growth, the bacteria only express BADE protein but not lysis protein, when the bacteria reach stationary phase, the lysis protein gene will be induced expression, due to the toxicity of lysis protein, engineering bacteria lysis.This is the basis for our design of the self-lysis function of Bacillus subtilis.
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Detection of self-lysis function
Since the concentration of bacterial solution is positively correlated with its absorbance at 600nm, the effect of lysis is evaluated by measuring the absorbance of cultured bacterial solution.
We designed two protocols to integrate each component
1. The aflatoxin degradation enzyme BADE was conjured to the P43 promoter, because P43, as an integrated promoter in Bacillus subtilis, does not require inducer compared with T7 and lactose operon, thus making it safer to add to 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（Fig. 1）. 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.
Figure 1. Four combinations
2. According to the investigation, pAX01 plasmid can be integrated into the genome of Bacillus subtilis. Therefore, four combinations of lysate protein and lysate protein promoter from part 1 can be cloned into pAX01 plasmid, which can be integrated into the genome of Bacillus subtilis after transfer. 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.
3. The selenium enrichment of Bacillus subtilis is innate.
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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
