1、 The fusion gene sequence was obtained
According to the relevant data of NCBI, we did not find the sequence of merR and merB in Bacillus subtilis, but we found the corresponding sequence in Bacillus megaterium. Considering the high homology between Bacillus subtilis and Bacillus megaterium, we plan to design primers based on the gene sequence of Bacillus megaterium, and try to amplify the corresponding genes in Bacillus subtilis (specific For information, see: parts1), cloned from Bacillus megaterium
We intend to fuse merR with terminator sequence and PmerR-merB3 with atLCD sequence. For this purpose, we designed the primers needed for fusion PCR: (see parts2 for details)
2、 Construction and replication of plasmid
We used the shuttle plasmid pAX01 of Bacillus subtilis as the vector to construct each element. pAX01 plasmid had three restriction sites (SpeI、BamHI、SacII) in the insertion region (see Part 3 for details of plasmids). We also added the corresponding restriction sites on the upstream and downstream primers of the fusion sequence. The fusion gene obtained by fusion PCR was inserted into plasmid by double enzyme digestion.
The obtained plasmid was introduced into competent E.coli and cultured in the medium containing ampicillin antibiotics (see Part 3, the plasmid has ampicillin resistance), and multiple copies of our target plasmid were obtained. The target plasmid was amplified by PCR using Check-F and check-R sequences as primers, and DNA electrophoresis was performed to verify the connection. If the electrophoresis results show that the size of the band is consistent with the sum of the two fusion genes, it is proved that the connection is successful and sent to the gene company for sequencing confirmation.
Figure 1: schematic diagram of components: merR—Ter—pMerR--MerB3—RBS—atLCD
3、 Transformation of Bacillus subtilis
The target plasmid was extracted from E.coli by plasmid extraction kit and transferred into Bacillus subtilis. (see Part 3, the plasmid is shuttle plasmid). The transformed bacterial solution was coated on the plate medium containing erythromycin (erm) antibiotics, and the transformed strains were screened and expanded. (see Part 3, which is resistant to ampicillin)
4、Validation of expression effect
As described in part 3, our plasmid contains xylose operon elements, which can efficiently express the target gene under xylose induced conditions. Bacillus subtilis was induced to express in the presence of xylose. The engineering bacteria were divided into two bottles of culture medium, one bottle was not added with mercury ion, the other bottle was sent to the analysis and testing center, added 0.2 mM mercuric chloride, and cultured in shaking table for 24 h. After the expansion of culture, two bottles of bacterial liquid were sampled respectively, and 13000 were centrifuged to obtain the bacterial precipitation. (the operation of mercury containing medium is completed by the analysis and test center) PBS suspension was used to crush the cells. The supernatant was centrifuged and the precipitate was centrifuged for protein electrophoresis to verify the protein expression. According to the expectation, MerB3 and AtLCD proteins should not be expressed or very low expressed in the culture medium without adding mercury ions, while MerB3 and AtLCD should be highly expressed in the medium supplemented with mercury ions. (if the soluble expression of the protein is poor, we should consider optimizing the induction conditions, or using a weaker plasmid promoter for the experiment.)
5、 Verification of mercury pollution control ability of engineering bacteria
The obtained engineering bacteria were sent to the analysis and testing center. 0.2 mm mercury chloride and 1.0 ppm methyl mercury chloride were added into the culture medium of the engineering bacteria. The culture medium with 30 min, 1 h, 3 h, 6 h and 12 h was taken respectively. After the bacteria liquid was removed by centrifugation, the residual mercury ion and organic mercury content were determined. It is expected that the content of mercury ion and organic mercury in the culture medium can be significantly reduced by our engineering bacteria, and the residual mercury content will gradually decrease with the growth of culture time.
If the experimental results are in line with the expectation, the relevant departments in the areas with high mercury content, such as Wanshan mining area in Guizhou Province, can be contacted to obtain local soil samples. The engineering bacteria (treatment 1) and engineering bacteria and earthworms (treatment 2) are added to the actual soil samples. The above experiments are repeated to determine the mercury content in the soil before and after treatment and the change of mercury content with time. It is expected that the experimental results are consistent with the above.(See model for treatment 1 and treatment 2).
In our design, we expect to construct engineering bacteria that can express merR, BFP, atlcd, merB3 and metallothionein, and hope to coordinate the expression of each protein by designing a signal amplification circuit, in order to achieve the most efficient the result of. However, in order to ensure the maximum feasibility of the experiment, we decided to construct engineering bacteria that only express merR, atLCD and merB in the previous experiment, and by using high expression vectors, the steps of designing the signal amplification circuit were simplified. Promoter to achieve signal amplification. In our preliminary experiments, we hope to verify whether the core mercury fixing element composed of merR, merB and atLCD can function as expected. If the results of the experiment are successful, we will add BFP and metallothionein in subsequent experiments to further improve the functions of the engineered bacteria and regulate the protein expression to achieve the synergy of different proteins.
JUVENILE'S ACTIVITIES CENTRE OF XUHUI
8 Tianlin East Road, Shanghai,China
GET IN TOUCH