Qualitative test

Experimental background

Based on the genomic, metabonomic, transcriptome and proteomic data obtained in the laboratory, the metabolic pathways and differential metabolites, differentially expressed genes and differentially expressed proteins in the normal environment (NaCl:10g/L, pH ±7) and high saline-alkali ring (NaCl:10g/L, pH=10) of six strains of saline-alkali-tolerant bacteria were analyzed. Or the saline-alkali tolerance gene elements shared by Gram-positive bacteria and Gram-negative bacteria respectively, and then qualitatively verify the screened gene elements in order to find universal functional gene elements. The main research contents are qualitative verification: in-depth analysis of candidate gene elements, selection of expression vectors and host expression bacteria, construction of recombinant expression plasmids, transduction of recombinant plasmids and determinatio n of saline-alkali tolerance of recombinant engineering bacteria; to determine whether the candidate gene element has saline-alkali tolerance.

1.Experiment One

Brief introduction of experiment

Through codon optimization, the target gene elements to be verified were selected for heterologous overexpression in Escherichia coli, and the saline-alkali tolerance of recombinant Escherichia coli was tested. the salt-alkali tolerance function of candidate gene elements has been determined.

1.1 Construction and verification of plasmid

1.1.1 Construction of recombinant plasmid

We selected Holomonas campaniensis LS21 selected from the soil of Lake Aidin, and its habitat is sodium salt environment. GenBank entry number: PRJNA248383. The selected vector is an expression vector pET28a(+) plasmid sequence of E. coli.

The Optimized (for Escherichia coli) sequence of LS21sufB

A 492 T 374 C 253 G 336 | GC%: 40.48% | Length: 1455

Construction of recombinant plasmid with 5’Noc I、3‘Hind III as restriction sites

1.1.2 restriction enzyme digestion experiment to verify the construction of plasmid

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.

1.2 Preparation of competent cells of Escherichia coli

1.3 Transformation of plasmids

The constructed plasmid was added to the prepared competent cells. after treatment, the treated solution was coated on the plate, and the colony growth on the plate was observed. In order to verify whether the plasmid was transformed into the cell, the cells with the target plasmid were lysed, the target plasmid was extracted, and the extracted plasmid was digested with restriction endonuclease, and the results were detected by agarose gel electrophoresis.

1.4 screening of positive clones

The overnight colonies (2 parallel on each plate) were picked up into LB liquid culture medium containing 100 μ g / ml Kana at 37 ℃, 150rpm, overnight culture, seed solution was prepared, PCR verification was carried out.

1.5 Expression of target gene

The positive clones were activated and cultured, then 0.1mM IPTG was added and grown at 30 °C for 2 hours. The target protein was induced to express in the strain, then diluted with LB and inoculated with 50 μ l inoculum on plates to observe the growth every 12 hours.

1.6 Protein detection

1.7 Shake flask test of salt concentration of E coli.Dh5 α-pET-28a (+)-LS21sufB

Step: inoculate the induced bacterial liquid with 1% inoculum to the 250ml conical bottle (sample 100ml)

Control group: a wild type Dh5 α (WT) culture medium without kana, B empty load (EV) medium with kana. Experimental group: E coli. Dh5 α-pET-28a (+)-LS21sufB (LSD) medium contains Kana.

Note: the effect of adding antibiotic Carna to no-load and experimental medium is to prevent contamination of miscellaneous bacteria.

1.8 Shake flask test of E coli. Dh5 α-pET-28a (+)-LS21sufB base concentration

Step: inoculate the induced bacterial liquid with 1% inoculum to the 250ml conical bottle (sample 100ml)

Control group: a wild type Dh5 α (WWT) culture medium without Kana, B unloaded (EV) medium with Kana. Experimental group: e coli. Dh5 α-pET-28a (+)-LS21sufB (LsD) culture medium includes Kana .

Note: the effect of adding antibiotic Carna to no-load and experimental medium is to prevent contamination of miscellaneous bacteria.

1.9 Determination of saline-alkali tolerance of LS21sufB engineering 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.

Results:

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.

2.Experiment 2

Collection and test of strains

We collected six strains of salinity-tolerant bacteria from dung beetles' intestinal tracts, poplar trees, uranium mines, salt lakes, and saline fields. (Brevibacterium casei G20, Bacillus haynesii P19, Micrococcus luteus R17, Halomonas bluephagenesis TD01, Halomonas campaniensis LS21 and Enterobactercloacae RS3），

We observed the growth condition of six strains in different saline-alkaline environment. Then，we did gradient saline-alkaline tolerance assay, saline-alkaline decoupling and coupling tests, saline tolerance extremes assay and made the growth curve.

The overall experimental process from objective gene to genetically engineered bacteria:

Nautural strains collection → tolerance test → saline-alkaline decoupling tests → saline-alkaline coupling tests → selection

Here are some details about our experiments.

1.Single colony morphology

Fig 1 shows the difference in single colony morphology of the six strains under normal and high saline and alkaline conditions(pH=7.0、10g/L NaCl and pH=10.0、60g/L NaCl).

These results demonstrate ability of every strains to tolerate saline-alkaline environments , and lay the foundation for the next experiment. This experiment not only help us to understand the differences in the strains' saline-tolerance ability accurately but do good to the selection for future industrial utilization.

2. Salt and alkali decoupling and coupling tests

The growth of six strains of salinity-tolerant bacteria under salt and base decoupling and coupling conditions (pH=7.0, 10g/L NaCl concentration, pH=7.0, 60g/L NaCl concentration, pH=10.0, 10g/L NaCl concentration, and pH=10.0, 10g/L NaCl concentration) is shown in Fig 2

Salt-alkali coupling results showed that RS35 strain shows poor performance in tolerating high salt and alkali, while G20, P19, R17, TD01 and LS21 strains perform well in same situation.

3. Gradient saline environment

The growth of six strains in gradient saline-alkali conditions are shown in Fig 2.3。（pH=7.0、8.0、9.0、10.0 and 10g/L、30g/L、60g/L、90g/L NaCl）

With the increase of salinity gradient, the colony morphology changed to different degrees, mainly in the form of higher transparency, lighter colour, slower growth rate and smaller colonies. The heat map of strains under different salinity gradients for 48h (Fig 3) shows that the growth of the strains was inhibited with the increase of salinity gradient. They grew slowly and their biomass reduced under high saline conditions. Additionally, in the same saline environment, different saline tolerant strains show differences in growth.

4. Salt and alkali tolerance limit test

The results of the salinity tolerance limits of the six strains are shown in Fig2.6.

The best salt-tolerant strain is H. bluephagenesis TD01, reaching 18%, and the best alkali-tolerant strain was found to be M. luteusR17, reaching pH=13.0.

Based on this, pH=7.0, 10g/L NaCl and pH=10.0, 60g/L NaCl were selected as experimental conditions for the later studies.