Microbiota reconstruction flowpot assay

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assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md

+## Microbiota reconstitution in the gnotobiotic Flowpot system
+
+Flowpot assembly was performed according to (24) with minor adjustments. A 2:1 mixture of peat potting mix and vermiculite was used as a matrix. The matrix was sterilized two times (25 min liquid cycle (121 °C) and 45 min solid cycle (134 °C)) and stored at 60 °C until completely dry. Prior to Flowpot assembly, the matrix was rehydrated with sterile MiliQ water. Flowpots were assembled by adding a layer of glass beads to the conical end of a truncated syringe, followed by a layer of the rehydrated, sterile substrate, subsequently covered with a sterile mesh secured by a cable tie. Assembled Flowpots were sterilized on a 25 min liquid cycle, stored at 60 °C overnight and sterilized twice on a 45 min solid cycle. Bacterial strains, cultivated as described before, were harvested, 3x washed and pooled in equal ratios. Then, 1.25 ml bacterial pool (OD~600~ 1.0) were added to 500 ml of 1⁄2 MS medium for a final bacterial OD~600~ of 0.0025. Flowpots were first flushed with sterile MiliQ water, then inoculated with 50 ml of 1⁄2 MS. Eight inoculated Flowpots were placed into each sterile microbox (TP1200, Sac O2) and stored at room temperature overnight. Exactly five sterilized seeds were sown per Flowpot and left to grow under the previously described  conditions. At 21 dpi, shoot fresh weight was measured individually for each plant. Roots from a single Flowpot were thoroughly cleaned from soil particles in sterile water using tweezers. Six representative Flowpots were selected for harvesting root and matrix samples. Cleaned roots from each Flowpot were pooled in 2 ml lysing matrix E tubes (MP Biomedicals), snap-frozen and stored at -80 °C until further use. Additionally, <100 mg of soil were taken from each Flowpot, snap-frozen and stored in weighed 2 ml lysing matrix E tube (MP Biomedicals) at -80 °C until further use.
+
+(24) J. M. Kremer, et al., Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research. *Nature Protocols 2021 16:5* **16**, 2450–2470 (2021).
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