This work was financially supported by the National Natural Science Foundation of China (42307173 and 42107328) and the National Key Research and Development Program (2022YFD1500202 and 2022YFF1001800). P.W, Z.X designed the study. P.W, B.X, Z.C, J.X, and N.Z analyzed the data and created the figures. P.W wrote the first draft of the manuscript. Z.X., R.Z., and Q.S. revised the manuscript.

This protocol is generally applicable to rhizosphere soil microbiota from various plants. Here, cucumber is used as an example. Details of the reagents and equipment used for the study are listed in the Table of Materials.
1. Bacterial isolation
<ol>
	<li>Rhizosphere soil isolation
	<ol>
		<li>Cucumber culture
		<ol>
			<li>Disinfect cucumber seeds with 75% ethanol and 2% sodium hypochlorite (NaClO) solution, then rinse them in sterile water11.</li>
			<li>Germinate the seeds under sterile conditions and select those that reach the two-leaf stage12. Subsequently, transplant the consistently sprouted cucumber seedlings into pots containing 2 kg of black soil. 
			NOTE: Two types of black soil from different locations in Northeast China are used to reduce systematic errors.</li>
		</ol>
		</li>
		<li>Soil suspension preparation
		<ol>
			<li>Prepare PBS-S buffer using the formulation: 6.33 g of NaH2PO4&#183;H2O, 16.5 g of Na2HPO4&#183;7H2O, 200 &#181;L of Silwet L-77, and 1 L distilled water.</li>
			<li>When the seedlings reach the four-leaf stage12, invert the pot and isolate the roots along with 2 mm-thick rhizosphere soil using sterilized gloves.</li>
			<li>Place the roots in 30 mL of PBS-S buffer in a 50 mL centrifuge tube. After shaking for 20 min, filter the suspension through a 100 &#181;m nylon cell filter to remove roots and large sediments.</li>
			<li>Transfer the filtrate to another 50 mL centrifuge tube, centrifuge it at 3200 x g for 15 min at room temperature, and temporarily store it at 4 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extraction of rhizosphere soil bacterial cells. 
	NOTE: This methodology adopts the differential centrifugation method13, although the Nycodenz density gradient centrifugation method14 also applies for this step.
	<ol>
		<li>Dilute the soil suspension with a 0.2% Na4P2O7 solution to 500 mL and centrifuge at 1,000 x g for 10 min to initially separate organisms from the soil.</li>
		<li>Re-homogenize the precipitate, centrifuge it at the same low speed for 60 s, and repeat the process three times. Combine the supernatants, centrifuge them at 12,000 x g for 20 min, and then discard the supernatant.</li>
		<li>Resuspend the precipitate in 50 mL of PBS-S buffer, constituting the rhizosphere soil bacterial cell suspension.</li>
	</ol>
	</li>
</ol>
2. Sequencing and identification of rhizosphere biofilm microbiota
<ol>
	<li>Media preparation 
	NOTE: Tryptic soy broth (TSB) and Minimal salts glycerol glutamate (MSgg) media are commercially available. The formulations below are for reference only.
	<ol>
		<li>Prepare TSB medium using the following formulation: 15 &#181;g/mL tryptone, 5 &#181;g/mL soy peptone, 5 &#181;g/mL NaCl, pH = 7.</li>
		<li>Prepare MSgg medium using the following formulation: 5 mM KH2PO4, 100 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 2 mM MgCl2, 700 &#181;M CaCl2, 50 &#181;M MnCl2, 50 &#181;M FeCl3, 1 &#181;M ZnCl2, 2 &#181;M thiamine, 0.5% glycerol, 0.5% glutamate, 50 &#181;g/mL tryptophan, 50 &#181;g/mL phenylalanine, pH = 7.</li>
		<li>To prepare TSB-MSgg media, mix TSB media with MSgg media in a 1:1 ratio (v/v)15.</li>
	</ol>
	</li>
	<li>Pellicle biofilm coculture
	<ol>
		<li>Incubate the cell suspension in TSB medium at 30 &#176;C overnight (usually 12 h) to activate the bacteria.</li>
		<li>Place 100 &#181;m nylon cell filters on 24-well plates. Incubate the bacteria in TSB media and set up three repetitions. Cultivate the pellicle biofilm for 36 h at 30 &#176;C. 
		NOTE: If the pellicle biofilm from the last step is not well-cultivated, the TSB-MSgg medium is an effective substitute for the TSB medium.</li>
		<li>Remove the filter containing the pellicle biofilm. Add 2 mL of PBS-S buffer to a new 24-well cell plate, place the 24-well filter into it to wash the biofilm, and remove the free cells. Repeat the washing process three times.</li>
	</ol>
	</li>
	<li>Extraction of pellicle biofilm DNA
	<ol>
		<li>Extract the biofilm genome using a DNA kit following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>High-throughput sequencing 
	NOTE: Biotech companies can help complete sequencing experiments for most labs. If microbial composition in the remaining solution is needed, sequence it at this step. The sequencing step varies depending on the instruments. Please follow the manufacturer&#39;s instructions. Step 2.4.1 is for reference only.
	<ol>
		<li>Amplify the V3-V4 regions of the 16S rRNA gene based on the database with full-length 16S rRNA gene sequences. Create species OTUs according to the minimum number of sequences per sample16. Cluster the species classification annotations and acquire the compositions of communities from each sample.</li>
		<li>Based on the sequencing data, select the top five strains in terms of their relative abundance.</li>
	</ol>
	</li>
	<li>High-throughput bacterial isolation and culture 
	NOTE: This step is designed according to the latest microbiological and bioinformatic methodology17. The dilution-coated plate method is available for bacterial isolation here, although it is more time-consuming and less targeted compared to high-throughput techniques.
	<ol>
		<li>Mix the cultivated biofilm. Use limiting dilution to ensure that no more than 30% of the wells in the 24-well plate show bacterial growth. 
		NOTE: To determine the most suitable dilution, perform preliminary experiments using a wide range of dilution rates. The optimal dilution should contain no more than two culturable bacteria per milliliter, representing bacterial incubation in less than 30% of the wells.</li>
		<li>Amplify the 16S rRNA gene of the bacteria while adding labels for the wells and plates and sequence them using the high-throughput Illumina platform17.</li>
		<li>Conduct bioinformatic analyses using commercially available software to obtain purity and species taxonomic information for each culture.</li>
		<li>Cultivate the top five abundant pure bacteria by continuous line culture and use glycerol to preserve the bacteria at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Construction of synthetic microbial communities
<ol>
	<li>Re-constructed biofilm culture 
	NOTE: For details, please refer to Ren et al.18. The 5 strains correspond to 31 synthetic combinations, including 5 single-species, 10 two-species, 10 three-species, 5 four-species, and 1 five-species consortia.
	<ol>
		<li>Incubate the 5 strains in the TSB medium until their OD600 reaches 1 to activate them.</li>
		<li>Prepare several 24-well plates with TSB medium and place 100 &#181;m nylon cell filters on them. 
		NOTE: If the biofilm from the last step is not well-cultivated, the TSB-MSgg medium is an effective substitute for the TSB medium.</li>
		<li>Incubate the 31 combinations of synthetic communities in the medium. Ensure the inoculation number of bacteria is consistent in each well. Set up three repetitions for each combination.</li>
		<li>Cultivate the biofilm at 30 &#176;C for 36 h.</li>
	</ol>
	</li>
	<li>Quantification of pellicle biofilm
	<ol>
		<li>Follow the same procedure as in step 2.2.3.</li>
		<li>Transfer the biofilm to a new 24-well plate containing 180 &#181;L of crystal violet solution and stain it for 20 min.</li>
		<li>Transfer the stained sample to another 24-well plate containing 200 &#181;L of 96% ethanol, elute it for 30 min, and measure OD590.</li>
	</ol>
	</li>
</ol>
4. Pellicle cell number quantification
NOTE: To determine the proportion of each species and the cell counts in the pellicle and remaining solution, quantitative PCR is necessary. For details, refer to Sun et al.16.
<ol>
	<li>Design strain-specific primers for the selected synthetic microbial communities.</li>
	<li>Use Roary to perform genome comparisons and find out the strain-specific single-copy genes of each isolate. Ensure that primers are targeted to these genes.</li>
	<li>Ligase the amplified fragments to PMD19T plasmids. Generate standard curves using the plasmids containing corresponding fragments as templates.</li>
	<li>Follow the same steps as mentioned in step 2.2.</li>
	<li>Prepare reaction components as follows: 7.2 &#181;L of H2O, 10 &#181;L of 2x qPCR Master mix, 0.4 &#181;L of 10 &#181;M of each primer, and 2 &#181;L template DNA.</li>
	<li>Perform the PCR with a Real-time PCR Instrument under the following conditions: 95 &#176;C for 10 min, 40 cycles of 95 &#176;C for 30 s, and 60 &#176;C for 45 s, followed by a standard melting curve segment.</li>
	<li>Perform six biological replicates for each treatment.</li>
</ol>

Developing Synergistic Multispecies Biofilms from Rhizosphere Soil

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-growth-promoting+rhizobacteria.">Plant-growth-promoting rhizobacteria.</a> Annu Rev Microbiol. 63, 541-556 (2009).</li><li>Compant, S., Cl&#233;ment, C., Sessitsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+growth-promoting+bacteria+in+the+rhizo-+and+endosphere+of+plants:+Their+role,+colonization,+mechanisms+involved+and+prospects+for+utilization.">Plant growth-promoting bacteria in the rhizo- and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization.</a> Soil Biol Biochem. 42, 669-678 (2010).</li><li>Bais, H. P., Fall, R. R., Vivanco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocontrol+of+Bacillus+subtilis+against+infection+of+Arabidopsis+roots+by+Pseudomonas+syringae+is+facilitated+by+biofilm+formation+and+surfactin+production.">Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production.</a> Plant Physiol. 134 (1), 307-319 (2004).</li><li>Trivedi, P., Leach, J. E., Tringe, S. G., Sa, T., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-microbiome+interactions:+From+community+assembly+to+plant+health.">Plant-microbiome interactions: From community assembly to plant health.</a> Nat Rev Microbiol. 18, 607-621 (2020).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+velezensis+stimulates+resident+rhizosphere+Pseudomonas+stutzeri+for+plant+health+through+metabolic+interactions.">Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions.</a> ISME J. 16, 774-787 (2021).</li><li>Pandhal, J., Noirel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microbial+ecosystems+for+biotechnology.">Synthetic microbial ecosystems for biotechnology.</a> Biotechnol Lett. 36, 1141-1151 (2014).</li><li>Grosskopf, T., Soyer, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microbial+communities.">Synthetic microbial communities.</a> Curr Opin Microbiol. 18, 72-77 (2014).</li><li>Lundberg, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+core+Arabidopsis+thaliana.+root+microbiome.">Defining the core Arabidopsis thaliana. root microbiome.</a> Nature. 488, 86-90 (2012).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+overlap+of+the+Arabidopsis+leaf+and+root+microbiota.">Functional overlap of the Arabidopsis leaf and root microbiota.</a> Nature. 528, 364-369 (2015).</li><li>Bakken, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+purification+of+bacteria+from+soil.">Separation and purification of bacteria from soil.</a> Appl Environ Microbiol. 49 (6), 1482-1487 (1985).</li><li>Yang, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell+extraction+from+fresh+and+stored+soil+samples:+Impact+on+microbial+viability+and+community+compositions.">Direct cell extraction from fresh and stored soil samples: Impact on microbial viability and community compositions.</a> Soil Biol Biochem. 155, 108178 (2021).</li><li>Ren, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+multispecies+biofilm+formation+and+quantitative+PCR-based+assessment+of+individual+species+proportions,+useful+for+exploring+interspecific+bacterial+interactions.">High-throughput screening of multispecies biofilm formation and quantitative PCR-based assessment of individual species proportions, useful for exploring interspecific bacterial interactions.</a> Microb Ecol. 68, 146-154 (2013).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+interactions+affect+the+biomass+of+synthetic+bacterial+biofilm+communities.">Metabolic interactions affect the biomass of synthetic bacterial biofilm communities.</a> mSystems. 8, e01045-e01123 (2023).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cultivation+and+identification+of+bacteria+from+the+plant+root+microbiota.">High-throughput cultivation and identification of bacteria from the plant root microbiota.</a> Nat Protoc. 16, 988-1012 (2021).</li><li>Ren, D., Madsen, J. S., S&#248;rensen, S. J., Burnolle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+biofilm+synergy+among+bacterial+soil+isolates+in+cocultures+indicates+bacterial+interspecific+cooperation.">High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation.</a> ISME J. 9, 81-89 (2015).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+colonization+by+beneficial+rhizobacteria.">Root colonization by beneficial rhizobacteria.</a> FEMS Microbiol Rev. 48 (1), 066 (2024).</li><li>Palkov&#225;, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+microorganisms:+Laboratory+versus+nature.">Multicellular microorganisms: Laboratory versus nature.</a> EMBO Rep. 5, 470-476 (2004).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+communication+in+plant-microbe+beneficial+interactions:+a+toolbox+for+precise+management+of+beneficial+microbes.">Chemical communication in plant-microbe beneficial interactions: a toolbox for precise management of beneficial microbes.</a> Curr Opin Microbiol. 72, 102269 (2023).</li></ol>

The authors have no conflicts of interest to disclose.

Following the protocol, a series of robust synthetic multispecies biofilm communities are constructed based on the microbiota in rhizosphere soil from different plants. Using quantitative PCR techniques, the composition of each community is deciphered clearly. The biomass of the biofilm indicates the strength of their potential metabolic interactions, although various properties and mechanisms behind the cooperation could not be revealed here19. Given the size and complexity of natural microbial c...

Biofilms represent intricate microbial communities either affixed to surfaces or linked with interfaces. There is broad acknowledgment that the majority of bacteria encountered in various environments, such as natural habitats, clinical settings, and industrial contexts, endure within biofilm communities1. In soil, the rhizosphere&#8212;encompassing the root surface and its adjacent 2 mm thick region&#8212;forms an ecological niche with heightened nutrient availability. Specific bacteria have evolved strategies to exploit this niche, fostering microbial proliferation and fostering the formation of biofilm communities in the rhizosphere2.
Rhizosphere microbes are considered the &#39;second genome&#39; of plants3. Plant growth-promoting microorganisms (PGPM) engage in mutualistic symbiosis with plants, providing significant growth promotion and biocontrol functions4. On the one hand, PGPM can supply nutrients to plant roots, enhance nutrient uptake, defend against pathogens, and degrade pollutants in the rhizosphere soil5. On the other hand, PGPM utilizes plant root exudates as a nutrient source for growth and evolution, thereby influencing the rhizosphere soil and root system through their own metabolism. It is worth noting that the prerequisite for all these functions is the effective colonization of PGPM in the plant rhizosphere, forming stable biofilms6.
Rhizosphere biofilm affects the assembly process of rhizosphere microbes, and the temporal and spatial characteristics of rhizosphere microbe assembly are closely related to the interaction of multispecies biofilm7. It serves as the hub of plant-microbiome interactions, providing a stable and controllable niche for them&#160;to interact effectively. Therefore, studying rhizosphere biofilm is also an important direction to gain insight into the interaction between plants and microbes.
However, there is currently little research on rhizosphere multispecies biofilms. The high complexity of microbiome composition makes it challenging to answer fundamental ecological questions surrounding natural microbial communities8. In the process of experiments, many conditions, such as soil type, plant type, and the large number of microbial communities, need to be considered. Various factors limit the research of rhizosphere multispecies biofilms.
To further investigate multispecies biofilm communities from rhizosphere soil, such as interspecies synergistic interactions or metabolite cross-feeding8, it is desirable to artificially construct a simplified, representative, stable, and tractable synthetic microbial community under laboratory conditions9,10. Here, a standardized protocol combines several of the latest microbiological and bioinformatic techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 % Na4P2O7 solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>20041418</td>
			<td>Used to dilute the soil suspension.</td>
		</tr>
		<tr>
			<td>100 &#956;m Nylon Cell Filter</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>F613463-0001</td>
			<td>Used to filter culture solution and separate pellicle biofilm.</td>
		</tr>
		<tr>
			<td>2% NaClO solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>L03336903</td>
			<td>Used to sterilize the seeds.</td>
		</tr>
		<tr>
			<td>24-well Plate</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>PSRP010</td>
			<td>Used for micobial cultivation and pellicle biofilm separation.</td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propane sulfonic acid (MOPS)</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2360010</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Beijing Su-bio Biotech Co., Ltd.</td>
			<td>62.547.254</td>
			<td>Properly-sized centrifuge tubes for our protocol.</td>
		</tr>
		<tr>
			<td>75% C2H5OH solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>801769680</td>
			<td>Used to sterilize the seeds.</td>
		</tr>
		<tr>
			<td>Acinetobacter baumannii XL-1</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>Bacillus velezensis SQR9</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>Bacteria DNA Kit</td>
			<td>Nanjing Dinsi Biotechnology Co.,Ltd.</td>
			<td>D3350020000K04U021</td>
			<td>Used to extract bacterial DNA.</td>
		</tr>
		<tr>
			<td>Black Soil I</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Black soil from Jilin Province.</td>
		</tr>
		<tr>
			<td>Black Soil II</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Black soil from Heilongjiang Province.</td>
		</tr>
		<tr>
			<td>Burkholderia cenocepacia XL-2</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10035048</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>G508009-0001</td>
			<td>High-speed centrifuge.</td>
		</tr>
		<tr>
			<td>ChamQ SYBR qPCR Master Mix</td>
			<td>Vazyme Biotech Co.,Ltd.</td>
			<td>Q311-02</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Clean Bench</td>
			<td>Suzhou Antai Airtech Co., Ltd.</td>
			<td>NB026143</td>
			<td>Used for aseptic operation.</td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>ShangHai CIMO Medical Instrument Co.,Ltd</td>
			<td>GNP-9080BS-<img alt="Equation 1" src="/files/ftp_upload/66926/66926eq01.jpg" style="margin: auto;" /></td>
			<td>Used for microbial cultivation.</td>
		</tr>
		<tr>
			<td>Cristal Violet Dye</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>A600331</td>
			<td>Used for pellicle biofilm staining and quantifivation.</td>
		</tr>
		<tr>
			<td>Cucumber Seed</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>&#34;Jinchun I&#34; cucumber seed.</td>
		</tr>
		<tr>
			<td>Culturome v 1.0</td>
			<td>The Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences</td>
			<td>N/A</td>
			<td>Used for high-throughput rhizosphere soil bacterial cultivation and identification</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10011918</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Hefei Midea Refrigerator Co.,Ltd.</td>
			<td>BCD-556WKPM(Q)</td>
			<td>Used for strain preservation.</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2180020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10010618</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Hydroponic Planting Basket</td>
			<td>Yulv Furniture Store</td>
			<td>6526262626</td>
			<td>Used for cucumber hydroponics.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10017618</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>7791186</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10400101</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Msgg Medium</td>
			<td>Shanghai Bioesn Biotechnology Co.,Ltd.</td>
			<td>BES20791KB</td>
			<td>Pellicle biofilm culture medium.</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>S9390-100G</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>NaCI</td>
			<td>Chinasun Specialty Products co., Ltd.</td>
			<td>HS0416</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;H2O</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>S9638-25G</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>PBS-S Buffer</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>E607008-0001</td>
			<td>Basic buffer for living tissues.</td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>RYON</td>
			<td>RT3486L005</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Beijing Labgic Technology Co.,Ltd.</td>
			<td>BS-1000-T</td>
			<td>Used for strain inoculation.</td>
		</tr>
		<tr>
			<td>PMD19T Plasmid</td>
			<td>Takara Biomedical Technology (Beijing) Co.,Ltd.</td>
			<td>D102A</td>
			<td>Used to generate qPCR standard curves.</td>
		</tr>
		<tr>
			<td>Pot</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>YHHWS2401</td>
			<td>Used for cucumber soil culture.</td>
		</tr>
		<tr>
			<td>Primer for qPCR</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>N/A</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Real-Time PCR Instrument</td>
			<td>Thermo Fisher Scientific (China) Co.,Ltd.</td>
			<td>4484073</td>
			<td>Used to perform qPCR on selected pellicle biofilm.</td>
		</tr>
		<tr>
			<td>Roary</td>
			<td>The Wellcome Trust SangerInstitute</td>
			<td>N/A</td>
			<td>Used to design primers of qPCR</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Shanghai Zhichu Instrument Co.,Ltd</td>
			<td>ZQTY-50S</td>
			<td>Used for solution mixing and microbial cultivation.</td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Cytiva Bio-technology(Hangzhou) Co., Ltd.</td>
			<td>SL77080596</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Soy peptone</td>
			<td>Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd</td>
			<td>HB8275</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shanghai Yidian Analysis Instrument Co.,Ltd.</td>
			<td>76713100010</td>
			<td>Used for pellicle biofilm cristal violet quantifivation.</td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>SW150302</td>
			<td>Used to wash the pellicle biofilm.</td>
		</tr>
		<tr>
			<td>Sterilized Nitrile Gloves</td>
			<td>Beijing Labgic Technology Co.,Ltd.</td>
			<td>223016852LLZA</td>
			<td>Used for basic experimental operations.</td>
		</tr>
		<tr>
			<td>Template DNA</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>N/A</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2180020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>LP0042B</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2190020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>TSB Medium</td>
			<td>Guangdong Huankai Microbial Sci. &#38; Tech. Co.,Ltd.</td>
			<td>024048</td>
			<td>Broad-spectrum bacterial medium.</td>
		</tr>
		<tr>
			<td>TSB-Msgg Meidium</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Mixed medium for pellicle biofilm culture with wider applicability.</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Nanjing Chemical Reagent Co.,Ltd</td>
			<td>C0310520123</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
	</tbody>
</table>

an approach to constructing multispecies biofilm communities from rhizosphere soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

Following the mentioned procedure, significant gradients in biofilm-forming capacity from cucumber rhizosphere soil microbiota are observed (Figure 1). Biofilm amplicon sequencing confirmed the species in the pellicle (Figure 2). Based on the heat mapping of sequencing data, the top five bacterial species whose abundance in the pellicle is larger than that in the remaining solution were selected (Figure 3). Here, a typical group of ...

Author Spotlight: Developing Synthetic Microbial Communities for Generating Second-Generation Biofertilizers

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

an-approach-to-constructing-multispecies-biofilm-communities-from

Research

JoVE Journal

Biology

624 Views.  Nanjing Agricultural University. A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

Video: Author Spotlight: Developing Synthetic Microbial Communities for Generating Second-Generation Biofertilizers

Biology of Microbial Communities  - Interview

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6.

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.