Collection of Microbial Samples from Extreme Environments in Xinjiang

Located in the northwest of China, Xinjiang boasts unique and diverse extreme environments. The samples collected this time consist of precious soil samples, water samples, camel milk samples, and fecal samples gathered and preserved by the postgraduate team led by Professor Yue Haitao during the third scientific expedition in Xinjiang, as well as on-site collections carried out by team members in typical areas near the university during their holidays.

The sampling sites include the desert area on the edge of the Tarim Basin with severe soil salinization, the glacial front of the Tianshan Mountains with alpine conditions, the vicinity of salt lakes in the Junggar Basin with extreme pH values, the thermal river basins in the Altai Mountains with frequent geothermal activities, and the mud volcano areas with reductive chemical environments (such as the area near Aksu).

During the sampling process, we adopted corresponding sampling point layout and collection methods based on the characteristics of different samples. For soil samples, methods like the diagonal sampling point layout and the checkerboard sampling point layout were used, and sterilized sampling bags were employed to collect surface soil and sediments.

For water sample collection, sterile sampling bottles were used to avoid secondary pollution, and corresponding protective agents were added on-site to stabilize the microbial community. Camel milk samples and animal fecal samples were sealed and stored at low temperature using sterile centrifuge tubes or sampling bags, and every effort was made to complete the transportation in the shortest possible time.

While sampling, we recorded key environmental parameters such as temperature, pH value, and salinity in detail. After collection, all samples were quickly placed in portable low-temperature boxes, and upon being brought back to the university, they were immediately transferred to a -80°C ultra-low temperature refrigerator for storage, so as to ensure the integrity of microbial DNA and the reliability of subsequent analysis.

This project has also established a cooperative relationship with the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. With its support, the integration of sample resources and the complementarity of scientific research conditions have been realized, laying a solid foundation for the subsequent exploration of microbial resources in extreme environments and the research on functional genes.

Isolation and Purification of Strains

Sample Pretreatment

1. For soil samples collected from various regions in Xinjiang, weigh 1g of each soil sample and mix it with 10ml of PBST by shaking to prepare the initial dilution. Then, reduce the bacterial concentration to 10⁻⁴ through gradient dilution to prevent colony overlap in subsequent cultivation.

2. Water sample pretreatment: After collecting the water sample, shake it well first, and adjust the pH to ≤ 2 with acid as needed. Before detection, take out the sample to rewarm it, filter it through a 0.2μm filter membrane to remove suspended solids, separate impurities by centrifugation, and add reagents such as nitric acid to digest organic matter and release target substances. The water sample can be tested only after ensuring no interference.

3. Milk sample pretreatment: Take an appropriate amount of milk sample and perform gradient dilution with sterile normal saline.

4. After collecting fresh camel feces, quickly put them into a sterile container, add sterile PBS buffer or normal saline, and mix thoroughly. Centrifuge at 3000-5000×g at low temperature for 10-15 minutes to remove large granular impurities, and take the supernatant. The pretreated sample is subjected to gradient dilution and then spread on a selective medium for cultivation.

Selective Cultivation and Single Colony

Isolation

According to the needs of subsequent experiments, we prepare five types of solid media, namely TSB, R2A, 2216, actinomycete, and silicate media, for isolating target strains. The single colony isolation is mainly carried out by the spread plate method and the streak plate method:

• For the spread plate method: Take 100μl of the gradient-diluted bacterial solution and spread it evenly on the surface of the above five solid media. After cultivation, each viable bacterium in the bacterial solution will multiply to form a visible single colony.
• For the streak plate method: Adopt the three-zone streak method. Dip a small amount of the diluted bacterial solution with a sterile inoculating loop, streak within the divided zones, and finally obtain single colonies at the end of the streaks.

Purification

Pick the single colonies into a container with 8ml of liquid medium, and place it in a shaker at 37°C with 200 rotations per minute for expanded cultivation. Observe whether the morphology of the colonies after cultivation is uniform.

The qualified pure strains after verification are cryopreserved in glycerol tubes (-80°C for long-term preservation) to maintain the activity and genetic stability of the strains.

Key Notes on Terminology Translation

• PBST: Abbreviation of Phosphate Buffered Saline with Tween, a commonly used buffer in microbiological experiments, retaining the original abbreviation for consistency in academic contexts.
• Gradient dilution: Translated literally to conform to the standard expression in microbiology, referring to the stepwise dilution of a sample to reduce microbial concentration.
• Selective medium: A medium that inhibits the growth of unwanted microorganismsisms while promoting the growth of target strains, using the standard term in microbiological English literature.
• Glycerol tube: A container filled with glycerol solution used for cryopreservation of strains, retaining the concise and commonly used translation in laboratory settings.

Plasmid Extraction Experiment

The core principle of the plasmid extraction experiment is the alkaline lysis method. This method uses an alkaline solution with high pH (such as NaOH) to disrupt the cell wall and cell membrane, leading to bacterial cell lysis and the release of plasmid DNA and chromosomal DNA.

During the experiment, the Biomek i5 Workstation is mainly used for high-throughput plasmid extraction. In the high-throughput stage, it integrates the SPRI (Solid Phase Reversible Immobilization) magnetic bead purification technology.

The specific process of high-throughput extraction is as follows: After cell lysis and neutralization, the plasmid DNA in the supernatant specifically binds to paramagnetic beads in the presence of isopropanol. Through magnetic field separation, ethanol washing, and elution with a low-salt buffer, purified plasmids are finally obtained.

This high-throughput method is suitable for the rapid processing of large batches of samples, and the concentration and purity of samples can be directly detected using a microplate reader. In the experiment, three parallel samples are first set up; if there are inconsistencies in the data of parallel samples or doubts about the results during the high-throughput extraction process, the Tiangen kit is further used for manual extraction verification.

The manual extraction method adopts silica membrane column purification technology after alkaline lysis: Under high-salt conditions, plasmid DNA specifically adsorbs to the silica membrane. Impurities are removed through deproteinization and rinsing steps. Finally, pure plasmids are obtained by elution with a low-salt elution buffer, and the concentration, purity, and integrity of the plasmids are verified using a microplate reader and agarose gel electrophoresis.

The extracted plasmids were verified by agarose nucleic acid electrophoresis, and the results are shown in the figure2.

Lane Number	Plasmid Number	Lane Number	Plasmid Number	Lane Number	Plasmid Number
1	pRM 1	14	pRM 14	27	pEM 2
2	pRM 2	15	pRM 15	28	pEM 3
3	pRM 3	16	pRM 16	29	pWM 1
4	pRM 4	17	pRM 17	30	pWM 2
5	pRM 5	18	pRM 18	31	pKM1
6	pRM 6	19	pRM 19	32	pKM2
7	pRM 7	20	pSM 1	33	pKM3
8	pRM 8	21	pSM 2	34	pKM6
9	pRM 9	22	pSM 3	35	pCM1
10	pRM 10	23	pSM 4	36	pCM2
11	pRM 11	24	pSM 5	37	pCM3
12	pRM 12	25	pSM 6	38	pCM4
13	pRM 13	26	pEM 1

Stress Test on Natural Bacteria

To verify whether the natural plasmid-containing bacterial strains isolated by our team from the harsh environment of Xinjiang truly possess the toughness required for industrial applications, our team adopted a comprehensive multi-stress preliminary screening strategy during the robustness test.

We simulated common stress conditions in industrial fermentation, including high temperature (up to 45°C), high salt (5%-7% NaCl), alkalinity (pH 9.0–10.0), acidity (pH 3.5–5.0), and various other stress conditions, to conduct a preliminary tolerance assessment on the wild strains.

By observing their growth curves, colony morphology, and survival rates, we initially determined under which types of stress the strains exhibit strong natural resistance.

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.

The activated seed culture of the wild strain was cultured with shaking at 37°C and 200 rpm until the OD₆₀₀ reached approximately 0.8, and then inoculated at a 1% inoculation amount into liquid media containing stress factors.

Similarly, 100 μL of bacterial solution with different dilution factors was spread on the corresponding solid LB plates (supplemented with 1.5% agar and adjusted stress factors). Three biological replicates were set up for each group.

During the culture period, samples were taken every 12 hours, and the OD value of the liquid culture was measured at a wavelength of 600 nm using a microplate reader to plot the growth curve.

For the solid plates, the morphology, size, and quantity of colonies were observed and recorded every 24 hours.

For high-temperature stress, a shaker with a temperature gradient from 40°C to 45°C was set up to compare the growth performance of the strains at different temperatures.

For low-temperature stress, a shaker with a temperature gradient of 16°C and 18°C was set up to compare their growth performance under different temperature conditions.

Establishment and Functional Verification of Recombinant Engineered Bacteria

Specific primers containing homologous arms were designed, and linearized recombinant fragments were obtained through PCR amplification.

Using seamless cloning technology, the reaction was carried out in a PCR instrument for 50 minutes. In vitro, the linear fragments were connected with linearized vectors through complementary pairing of homologous arms to form circular recombinant vectors.

The recombinant vectors were introduced into E. coli BL21 competent cells by the heat shock method (heat shock in a 42°C water bath for 90 seconds, followed by rapid cooling in an ice bath for 3 minutes).

After the transformed bacteria were resuscitated, they were spread on LB solid resistance plates containing kanamycin sulfate at a final concentration of 50 μg/mL for positive clone screening.

After picking single colonies for culture, the correctness of the recombinant vector construction was confirmed by two methods: colony PCR (to verify fragment insertion) and gene sequencing (to verify no sequence mutations).

A normal 1 L culture medium was prepared. Hydrochloric acid was added to the medium through a filter membrane to obtain an acidic medium, or sodium hydroxide stock solution was added to adjust the pH to obtain an alkaline medium.

300 μL of the engineered bacteria transformed with the natural plasmid was added to 30 mL of LB liquid medium for activation. When the activated strain grew to the late logarithmic growth phase, 1 mL of the bacterial solution was added to 100 mL of liquid LB medium containing Kana⁺, and cultured with shaking in a constant temperature shaker at 37°C and 150 rpm to draw the growth curve.

Wild-type Escherichia coli E. coli BL21 was used as the control group to detect the growth of wild-type and recombinant strains in the experimental environment. The growth performance of the strains was determined by the absorbance at OD₆₀₀ nm.

LB media under high-salt, acidic, alkaline, and normal conditions were prepared. For each condition, wild-type E. coli BL21 was used as the control, and 3 biological replicates were set up for each condition.

The strains were cultured at 37°C/16°C/18°C/45°C and 150 rpm, and their growth was observed. The OD value was measured every 12 hours.

ORF Prediction

The DNA sequence was analyzed using the ORF Finder tool in NCBI, combined with the analysis of TBtools software, to identify potential start and stop codons. The positions of ORFs (start-stop sites) and the corresponding amino acid sequences were output, thereby determining the functional regions that may encode proteins.

Some of the experimental results are shown in Figures 4.1.

Restriction Enzyme Verification

All sequences were optimized for codons based on the E. coli expression system to obtain the target sequence encoding the mature protein. After submitting the sequence to Youkang Biotechnology Co., Ltd. for chemical synthesis, the sequence was cloned into the pET-28a(+) vector. The recombinant plasmid was subjected to a double restriction enzyme reaction system, and double restriction enzyme verification was performed by reacting with ApaI-XhoI at 37°C for 3 hours.

Some of the experimental results are shown in Figures 4.2.

SDS-PAGE Analysis

Bacteria containing the recombinant plasmid were cultured in a stress environment corresponding to the plasmid function. When their growth OD₆₀₀ reached 0.8-1.0, the recombinant engineered bacteria were picked and inoculated into 30 mL of LB liquid medium containing 50 μg/mL Kana, followed by overnight culture at 37°C with 150 rpm to prepare the seed solution.

The seed solution was transferred to 50 mL of LB liquid medium containing Kana (50 μg/mL) at a 1% inoculation amount, and cultured at 37°C with 200-220 rpm until the OD₆₀₀ reached approximately 0.6-0.8. Then, induction was performed with 0.6 mM IPTG at 37°C for 6 hours.

The induced bacterial culture was centrifuged (4°C, 12000 rpm for 5 minutes), the supernatant was discarded, and PBS and 5× loading buffer were added at a ratio of 4:1, mixed well, and boiled at 100°C for 15 minutes. The supernatant was retained for subsequent SDS-PAGE analysis.

Some of the experimental results are shown in Figures 4.3.

Lane	Protein
Lane 1	Presumptive replication protein, uninduced
Lane 2	Presumptive replication protein, induced
Lane 3	Chromosome partitioning protein, uninduced
Lane 4	Chromosome partitioning protein, induced
Lane 5	Main partitioning protein parA, uninduced
Lane 6	Main partitioning protein parA, induced
Lane 7	Induced head protein, uninduced
Lane 8	Induced head protein, induced

Functional Verification

The procedure was the same as the functional verification step of the recombinant engineered bacteria.

Some of the experimental results are shown in Figures 4.5.

Experimental Equipment and Reagents

Experimental Equipment

Instrument/Equipment	Company
Portable High-Pressure Steam Sterilizer (DSX-30L)	Shanghai Shen'an Medical Instrument Factory
Microplate Reader (MULTISKAN)	Sky Thermor Company
Electric Constant-Temperature Incubator (DHP-360)	Beijing Yongguangming Medical Instrument Co., Ltd.
Constant-Temperature Incubation Shaker (ZHWY-211B)	Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.
Electronic Balance (AL104)	Mettler-Toledo Instrument Co., Ltd.
Ultra-Low Temperature Refrigerator (U410-86)	Eppendorf Company
Electrophoresis Apparatus (DYY-6C)	Beijing Liuyi Instrument Factory
Gel Imaging System (Gel Doc 2000 UV)	Bio-Rad, USA
Pipette (Research® plus)	Eppendorf, Germany
High-Speed Refrigerated Centrifuge (Heraeus Fresco21)	Heraeus Fresco
Constant-Temperature Water Bath (DK-8D)	Shanghai Qixin Scientific Instrument Co., Ltd.
Biomek i5 Automated Workstation	Beckman Coulter
pH meter（TESTO510）	Testo Company
clean bench（SW-CJ-2FD）	Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory

Experimental Materials

Experimental Reagent	Company
Tryptone	Beijing Aoboxing Biotechnology Co., Ltd.
Yeast Extract	Beijing Aoboxing Biotechnology Co., Ltd.
Sodium Chloride	Tianjin Sheng'ao Chemical Reagent Co., Ltd.
Sodium Hydroxide	Tianjin Sheng'ao Chemical Reagent Co., Ltd.
Agar Powder	Beijing Aoboxing Biotechnology Co., Ltd.
Non-Toxic Nucleic Acid Dye	Sangon Biotech (Shanghai) Co., Ltd.
Agarose	Sangon Biotech (Shanghai) Co., Ltd.
Yeast Powder	Beijing Aoboxing Biotechnology Co., Ltd.
2216E Liquid Medium	Qingdao Haibo Biotechnology Co., Ltd.
Actinomycete Medium (Modified Gao's No.1)	Qingdao Haibo Biotechnology Co., Ltd.
R2A Agar Medium	Qingdao Haibo Biotechnology Co., Ltd.
Tryptic Soy Broth Liquid Medium	Qingdao Haibo Biotechnology Co., Ltd.
Silicate Bacteria Medium (with agar)	Qingdao Haibo Biotechnology Co., Ltd.
Plasmid Mini-Prep Kit	Tiangen Biochemical Technology Co., Ltd.
One-Step PAGE Color Gel Ultra-Fast Preparation Kit

Reagent Preparation

LB Medium

Medium Component	Content (g/L)
Tryptone	10
Yeast Extract	5
Sodium Chloride	10

R2A Solid Medium

Medium Component	Content (g/L)
R2A Agar Medium	18.12

Actinomycete Solid Medium

Medium Component	Content (g/L)
Actinomycete Medium (Modified Gao's No.1)	37.5

2216E Solid Medium

Medium Component	Content (g/L)
2216E Liquid Medium	37.4
Agar Powder	40

Silicate Solid Medium

Medium Component	Content (g/L)
Silicate Bacteria Medium (with agar)	23.6

TSB Solid Medium

Medium Component	Content (g/L)
Tryptic Soy Broth Liquid Medium	30
Agar Powder	40

Agarose Gel

Gel Component	Content (g/L)
Agarose Powder	10

SDS-PAGE Gel Preparation

Using Servicebio's One-Step PAGE Color Gel Ultra-Fast Preparation Kit:

Operating Steps: