OVERVIEW

Relying on such an engineering model, we succeeded in obtaining universal gene originals in non-model strains that can improve the salinity and base resistance of model strains.

The experiment proves that our engineering is very successful, and such an experimental idea can also be used for mining other elements, we welcome every teams who want to do this work to communicate and use this method.

I will describe the method in detail and show the success we achieved in this page.

1. Tests of salinity and base resistance of wild bacterias

We collected wild strains from different habitats, purified them and tested for salinity tolerance. Six strains with good tolerance to salinity were obtained, and the results are shown in the figure.

2. Gene mining system

2.1 Method

To identify the common genetic elements differentially expressed in transcription, translation and metabolism that were commonly altered by the six salt-tolerant bacterial strains in response to salinity stress, we used p < 0.05 and |log2FC| > 0 as screening thresholds for the differentially expressed genes and differentially expressed proteins.

2.2 Result

There were two up-regulated differentially expressed genes, sufB (iron-sulfur cluster scaffolding complex subunit) and mnmA (tRNA-specific 2-thiouracilase), and no down-regulated differentially expressed genes among the six strains; there was one up-regulated differentially expressed protein, purM among the six strains.

The six strains of salinity-tolerant bacteria were screened for differentially expressed genes on both the transcriptome and proteome without differentiating between up- and down-regulation, and found that the six strains had 19 differentially expressed genes, the six strains had 21 differentially expressed proteins, and the six strains had five differentially expressed genes on both the transcriptome and proteome.（details are shown in model section）

In summary, a total of 40 genes with high likelihood were screened, and finally, in combination with the metabolomic data, the six most probable universal antisaline elements expressed at transcriptional, translational, and metabolic levels by saline-tolerant bacteria of different genera in response to different saline environments were listed. The results are as follows.

3．Construction of engineering bacteria

3.1 Plasmid construction

We chose T7 promoter and pet28a to construct recombinant plasmids. The pET-28a(+) vector and the target gene were double digested by Nco I and Hind III, and the gene was ligated to the vector using T4 ligase to obtain recombinant expression vectors of different sizes.

The following pictures show the recombinant plasmids we constructed and some of mappings

We have identified the surfB gene in five bacterial strains and have completed the detection of surfB. Here are the detailed plasmid mapping.

3.2 Plasmid verification

In order to verify whether the target gene was successfully constructed into the expression vector, the reaction system was digested at 37 ℃ for three hours, and then the results were detected by agarose gel electrophoresis (1% agarose gel (containing 0.5 μ g / mL nucleic acid dye), 90V electrophoresis for 30 minutes.

Results:

By observing the results of enzyme digestion, it was found that the band was consistent with the length of the target gene, which indicated that the target gene was successfully constructed into the expression vectorpET28a and could be transformed later.

3.3 Element effect detection

Before testing the effect of the components, we first ensured that the target gene surfB was successfully introduced into E. coli through experiments.

Results:

The constructed plasmid was added to the prepared competent cells, and after treatment, in order to verify whether the plasmid was transformed into the cells, the cells with the target plasmid were cleaved and then digested with endonuclease. The results of enzyme digestion were detected by agarose gel electrophoresis.

3.4 protein detection

4 Successful mining of components

Through the above steps, we have ensured that the target gene into imported and engineered bacteria successfully constructed. In the next experiment, we tested the changes of salinity tolerance ability of the engineered bacteria.

Engineering bacteria: e coli.BL21 (DE3)-pET-28a (+)-LS21sufB saline-alkali plate medium pH is 8.5.After induction, the bacterial solution is diluted to 10-2, and the inoculation amount in each cell is 30μL.

In order to verify the saline-alkali tolerance of LS21sufB engineering bacteria, four media with different sodium chloride gradients were cultured and observed. Compared with E coli.BL21 (DE3) wild-type, no-load and uninduced engineering bacteria, it was found that the engineering bacteria significantly improved the saline-alkali tolerance. The highest saline-alkali tolerance values of E coli.BL21 (DE3)-pET-28a (+)-LS21sufB were 10%NaCl and pH=8.5./p>