Design
Sampling was conducted in various harsh environmental areas of Xinjiang, followed by isolation and purification of the samples.
Plasmids were extracted using the alkaline lysis method, combining the use of an automated workstation with manual extraction verification.
Build
During sampling, corresponding layout and collection methods were employed based on sample characteristics. For soil samples, diagonal and checkerboard patterns were used with sterilized sampling bags for surface soil and sediment collection.
Water samples were collected using sterile sampling bottles to avoid contamination, with appropriate preservatives added onsite to stabilize microbial communities.
Camel milk and fecal samples were sealed in sterile centrifuge tubes or sampling bags and maintained at low temperatures to ensure rapid transfer.
The samples were diluted, and the bacterial strains in the resulting leachate were isolated using the dilution plating method. Single colonies grown were picked and subjected to monoclonal culture using the streak plate method.
16S rRNA gene sequencing was performed on the purified bacterial strains. Meanwhile, statistics were collected regarding the source, species, and quantity of the strains, followed by a comparative analysis of the strains' genera and species.
Test
A total of 2,000 bacterial strains were obtained through sampling, including 495 strains from milk samples, 182 strains from soil samples, 1078 strains from water samples, and 245 strains from fecal samples.
The collected samples were diluted to 10-4, and isolation was performed using five types of media: 2216E, Actinomycete medium, R2A, TSB, and Silicate medium, via the dilution plating method.
Single colonies were purified and cultured on LB medium using the streak plate method. Plasmids were extracted using an automated workstation.
Learn
After extraction, a total of 38 plasmid-containing bacteria were obtained, with a plasmid-containing rate of 1.9%. In the next step, the robustness of these natural plasmid-containing bacteria will be further determined.
Design
To verify the robustness of the natural plasmid-containing bacterial strains isolated from extreme environments in Xinjiang, our team adopted a comprehensive multi-stress preliminary screening strategy.
A preliminary tolerance assessment was conducted on the wild-type bacterial strains. By observing their growth curves, colony morphology, and survival rates, we initially determined the types of stress under which the strains exhibit strong natural resistance.
Build
We prepared culture media with different concentrations of NaCl (5%–7%), adjusted the pH to 9.0–10.0 using NaOH, and adjusted the pH to 3.5–5.0 using HCl.
For high-temperature stress, a shaking incubator with a temperature gradient of 40℃ to 45℃ was set up; for low-temperature stress, a shaking incubator with a temperature gradient of 16℃ and 18℃ was used to compare the growth performance of the strains at different temperatures.
The activated seed liquid of the wild-type bacterial strains was cultured with shaking at 37℃ and 200 rpm until the OD 600 reached approximately 0.8.
Test
After stress test cultivation under conditions of pH 4.2, pH 9.0, 6% NaCl, 45℃, and 18℃, we found that the bacterial strain exhibits acid resistance,alkali resistance,saline-alkali resistance,low-temperature resistance and high-temperature resistance function.
Learn
We have identified that 8 strains containing natural plasmids exhibit robustness.
To further explore the role of plasmids in these bacteria, we constructed recombinant engineered bacteria using the extracted plasmids and conducted tests on them.
Design
Natural plasmids were introduced into E. coli BL21 engineered bacteria, and stress environment experiments were conducted with blank engineered bacteria as controls.
The stress environment was the same as that used in the natural plasmid stress test.
Build
Before the experiment, reference sequences of the target gene family or entire genome were obtained from public databases (such as NCBI's GenBank, ENA, and UniProt). These sequences include the nucleic acid or protein sequences of the samples to be analyzed, as well as the sequences of known species used as references.
A local alignment database was constructed to compare the known plasmid sequences with plasmids of the same genus and species. Through comparative analysis of plasmid sequences, the conservation and specific differences of core functional modules (e.g., replicons, transfer elements) in their gene composition were revealed; the distribution and dynamics of functional elements such as resistance genes and mobile elements were identified. This comprehensive analysis was conducted to clarify the function, evolutionary strategy, and application value of the plasmids.
Specific primers with homologous arms were designed, and linearized recombinant fragments were obtained via PCR amplification. Subsequently, a seamless cloning kit was used, and the reaction was carried out in a PCR instrument for 50 minutes to connect the linear fragment with the linearized vector in vitro through complementary pairing of homologous arms, forming a circular recombinant vector.
The recombinant vector was introduced into E. coli BL21 competent cells via the heat shock method. After the transformed bacteria were resuscitated, they were spread on LB solid resistant plates containing kanamycin sulfate for positive clone screening. Single colonies were picked and cultured, and the correctness of the recombinant vector construction was confirmed by both colony PCR and gene sequencing.
Test
After stress test cultivation under conditions of pH 4.5, pH 9.0, 6% NaCl, 45℃, and 18℃, we found that the recombinant engineered bacteria containing pSM4,pSM8 and other plasmids exhibited alkali resistance, acid resistance, and saline-alkali function.
Learn
Through this round of experiments, we confirmed that plasmids play a role in enhancing the robustness of the host bacteria. Next, we conducted further verification to identify which specific fragments of the plasmids are responsible for this function.
Design
ORF (Open Reading Frame) prediction was performed on plasmids with robustness function. Recombinant engineered bacteria were constructed using ORFs with robustness function, followed by stress test cultivation and SDS-PAGE analysis.
Build
ORF prediction was performed to convert abstract DNA sequences into a specific list of functional genes, thereby systematically revealing the composition of plasmid replication and partitioning mechanisms, adaptive genes such as antibiotic resistance genes, and mobile elements like conjugative transfer elements.
All sequences were codon-optimized according to the E. coli expression system to obtain target sequences encoding mature proteins. The sequences were submitted to Youkang Biotechnology Co., Ltd. for chemical synthesis and then cloned into the pET-28a(+) vector. The recombinant plasmids were subjected to double enzyme digestion verification using an ApaI-XhoI double enzyme digestion reaction system at 37℃ for 3 hours.
The stress cultivation test was conducted in the same manner as that for the aforementioned recombinant engineered bacteria.
The bacteria containing the recombinant plasmids were cultured in a stress environment corresponding to the plasmid function. When the OD₆₀₀ of the bacterial culture reached 0.8–1.0, the recombinant engineered bacteria were picked and inoculated into 30 mL of LB liquid medium containing kanamycin (Kana), followed by overnight culture at 37℃ and 150 rpm to prepare the seed liquid.
The seed liquid was transferred to LB liquid medium containing Kana at an inoculation amount of 1%, and cultured at 37℃ with shaking at 200–220 rpm until the OD₆₀₀ reached approximately 0.6–0.8, then induced with IPTG.
The induced bacterial culture was centrifuged, the supernatant was discarded, and PBS and 5× loading buffer were added at a ratio of 4:1, mixed well, and boiled at 100℃ for 15 minutes. The supernatant was retained for subsequent SDS-PAGE analysis.
Test
Through this round of experiments, we confirmed that plasmids play a role in enhancing the robustness of the host bacteria.
Learn
Through this experiment, we found that ORF3 and ORF7 derived from pSM4, as well as ORF5 derived from pSM18, all exhibit tolerance under 6% NaCl and pH=9 environments.ORF12 shows tolerance under both pH=4.2 and pH=9 environments.