Ez rich medium

Manufactured by Teknova

EZ rich medium is a pre-mixed, ready-to-use culture medium designed for the growth and maintenance of a variety of bacterial strains. It provides essential nutrients and growth factors required for optimal cell growth and proliferation.

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Lab products found in correlation

Terrific broth medium, by Merck Group (1 mentions) Lyophilizer, by Labconco (1 mentions) Eclipse ti, by Nikon (1 mentions) Nhs cy3b, by GE Healthcare (1 mentions) C9100 13, by Hamamatsu Photonics (1 mentions) Zdc 2, by Olympus (1 mentions) Cellsens software, by Olympus (1 mentions) Ix83 inverted microscope, by Olympus (1 mentions) Kanamycin, by Merck Group (1 mentions) Carbenicillin, by Merck Group (1 mentions)

9 protocols using ez rich medium

Single-Molecule Imaging of E. coli LacY

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LacY-eYFP expressing E. coli C41 cells were grown overnight in LB medium with appropriate antibiotics and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.4% glycerol. When the cell culture reached an OD600 of 0.3, Lacy-eYFP expression was induced with 0.01%(v) L-arabinose for 30 minutes at 37 °C. The cells were centrifuged at 3000 rcf for 5 minutes and resuspended in EZ rich medium with 0.2% glucose. 3 μL of the bacterial culture was transferred onto a microscope coverslide and covered by an agarose gel pad.
Images were captured with an exposure time of 50 ms using the open source software μManager [92 (link), 93 ].
To obtain the final STORM reconstruction, 2000 frames were collected and processed with the ImageJ plugin ThunderSTORM [68 (link)].
All data and code used to generate the figures in this article can be found at Zenodo (DOI: 10.5281/zenodo.2637790) and GitHub, respectively.

Smit J.H., Li Y., Warszawik E.M., Herrmann A, & Cordes T. (2019). ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data. PLoS ONE, 14(6), e0217524.

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FFA Degradation and Production Assays

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For FFA degradation assays EZ Rich medium (Teknova) supplemented with 10 mM glucose was used. For FFA and FAMEs production assays either EZ Rich medium supplemented with 100 mM glycerol, Terrific Broth medium (EMD Millipore), or diluted sorghum hydrolysate was used. Sorghum hydrolysate was diluted 4x with M9 minimal salts medium to a final 1x M9 salts concentration. After preparation, all media were sterile filtered, not autoclaved, through a 0.22 μm bottle-top filter.

Valencia L.E., Incha M.R., Schmidt M., Pearson A.N., Thompson M.G., Roberts J.B., Mehling M., Yin K., Sun N., Oka A., Shih P.M., Blank L.M., Gladden J, & Keasling J.D. (2022). Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production. Communications Biology, 5, 1363.

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Anaerobic Fatty Acid Production in E. coli

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Anaerobic fatty acid production in E. coli DH5αZ1 transformed with pPJ176 (operons 1 and 2; Table 1) was compared to that of DH5αZ1 carrying the empty pBbA0k vector. For each strain, a 250-mL serum bottle containing 200 mL of EZ Rich medium (Teknova, Hollister, CA) supplemented with 0.2% glucose and 50 μg/mL kanamycin was inoculated from an overnight culture to a starting OD₆₀₀ of 0.005 and sealed with a butyl rubber stopper. The culture was incubated at 37°C shaking at 200 rpm until the OD₆₀₀ reached ~0.4, at which point gene expression was induced by addition of 200 nM anhydrotetracycline (ATc) under anaerobic conditions. Anaerobic growth was continued at 37°C overnight. The next day, cells were harvested in 30-mL high-strength glass centrifuge tubes and supernatant was decanted. The cell pellet was flash-frozen in liquid nitrogen and lyophilized overnight in a Labconco lyophilizer (Kansas City, MO). Biomass was stored at room temperature until fatty acid extraction.
The remaining operon strains were individually tested in the same manner, with the exception that cultures were grown in 125-mL serum bottles containing 100 mL media, and 3 mM KNO₃ was added under anaerobic conditions at the time of induction, where noted, to obtain higher cell density.

Javidpour P., Deutsch S., Mutalik V.K., Hillson N.J., Petzold C.J., Keasling J.D, & Beller H.R. (2016). Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli. PLoS ONE, 11(3), e0151087.

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Competitive Co-culture Assay of Auxotrophic Strains

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Separate culture tubes containing LB with chloramphenicol (25 µg/mL) were inoculated with ΔmetA or ΔpheA and incubated overnight at 37 °C with shaking. After 16 h, the cultures were diluted into 5 mL of EZ Rich Medium (Teknova) containing chloramphenicol (25 µg/mL) and 1 mM IPTG and lacking M and F at a final OD600 of 0.05. The initial ratio of ΔmetA to ΔpheA was 10 : 1, 1 : 1, or 1 : 10 (n = 3, for each starting ratio). The cultures were incubated at 37 °C with shaking for at least 24 h before transferring the community to fresh media using a 1 : 100 dilution. At this transfer time, the OD600 of each culture was measured and a 2 µL sample was spotted onto a glass slide for cell counting with microscopy (20× magnification) on a Nikon Eclipse Ti. Four images comprising four distinct fields of view were taken of each sample and each image was a composition of phase contrast, GFP and RFP channels. Subsequently, ImageJ was used to extract the number of ΔmetA cells from the GFP channel and ΔpheA cells from the RFP channel for each image.

Gupta S., Ross T.D., Gomez M.M., Grant J.L., Romero P.A, & Venturelli O.S. (2020). Investigating the dynamics of microbial consortia in spatially structured environments. Nature Communications, 11, 2418.

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Microfluidic Media Optimization for Cell Assays

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All plate reader and microscopy experiments were performed in EZ-rich medium (Teknova) prepared according to the manufacturer’s instructions with 0.2% glucose as the carbon source, except for the gating fidelity tests shown in Extended Data Fig. 8d,e, which were conducted in lysogeny broth (Difco). To prevent cell adhesion to microfluidic channels, all media were supplemented with 0.075% (w:v) Tween 20 (for consistency also in plate reader experiments). Selection antibiotics (50 μg ml⁻¹ of ampicillin, 50 μg ml⁻¹ of kanamycin and/or 34 μg ml⁻¹ of chloramphenicol) and basal inducers were added as required. A stock suspension of 1-μm-diameter fluorescent beads (Polysciences, Fluoresbrite Multifluorescent, catalogue no. 24062, visible in the RFP channel) was created at a ratio of one drop per 250 μl of MilliQ water. The resulting suspension was added to all microfluidic media at a dilution of 1:1,000 to be able to measure flow velocity in the media channels (see Microfluidics and microscopy). The specific AHL used in this study was N-(3-oxohexanoyl)-l-homoserine lactone, also known as 3-oxo-C6-HSL or Auto Inducer 1 for Vibrio fischeri. Large volumes of chemicals/inducers (for example, 5 M NaCl in MilliQ water used to reach a target concentration of 500 mM) were added by replacing the corresponding amount of water in the EZ-rich recipe.

Bittihn P., Didovyk A., Tsimring L.S, & Hasty J. (2020). Genetically engineered control of phenotypic structure in microbial colonies. Nature microbiology, 5(5), 697-705.

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Terpene Production from Recombinant E. coli

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The transformants containing each TPS gene were cultured in 15 mL of LB medium with 100 μg/L of ampicillin, 34 μg/L chloramphenicol, and 25 μg/L of kanamycin. The cultures were incubated at 37°C shaking at 220 rpm overnight. One mL of overnight culture was then inoculated into 20 mL of fresh EZ-rich medium (Teknova, CA) containing 20g/L glucose as well as the three aforementioned antibiotics and incubated at 37°C with shaking at 220 rpm until an OD_600nm of 0.8 was reached. Then terpene production was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) at the final concentration of 1mM and incubating for another 20 hours at 30°C with shaking at 180 rpm. Terpenes were extracted after 48 hours.

Wu W., Tran W., Taatjes C.A., Alonso-Gutierrez J., Lee T.S, & Gladden J.M. (2016). Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae. PLoS ONE, 11(2), e0146983.

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E. coli Labeling with Fluorescent Dye

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E. coli MG1655 cells were grown overnight in LB medium and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.2% glucose. The cells were grown until the culture reached an OD600 value of 0.3 after which the cells were centrifuged at 3000 rcf for 5 minutes and resuspended in PBS buffer. The amino-reactive dye Cy3B-NHS (GE Healthcare) was added to a final concentration of 1 mM and incubated while protected from light at room temperature for 1 hour [89 (link)]. Next, the cells were centrifuged at 3000 rcf for 10 minutes and the cell pellet was resuspended in PBS.

Smit J.H., Li Y., Warszawik E.M., Herrmann A, & Cordes T. (2019). ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data. PLoS ONE, 14(6), e0217524.

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eGFP Expression Monitoring in E. coli

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E. coli BL21(DE3) cells were grown overnight in LB medium and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.2% glucose. The growth media was supplemented with the appropriate antibiotics. When the cell culture reached an OD600 of 0.3, eGFP expression was induced with IPTG at a final concentration of 200 μM for 30 minutes at 37 °C.
For imaging, 3 μL of the bacterial culture was transferred to a microscope coverslip and covered by an agarose pad [90 (link), 91 (link)].
Imaging was done on an Olympus IX83 inverted microscope with an Olympus UAPON 100x NA 1.49 TIRF oil immersion objective. The excitation light (514 nm, Coherent) was coupled into the objective via the ET—442/514/561 Laser Triple band set (69904, Chroma) and the fluorescence was collected on a electron multiplying charge-coupled CCD camera (512x512 pixel, C9100-13, Hamamatsu). The focal position was held constant by the Olympus ZDC2 and images on multiple positions were collected automatically using Olympus’ CellSens software.

Smit J.H., Li Y., Warszawik E.M., Herrmann A, & Cordes T. (2019). ColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data. PLoS ONE, 14(6), e0217524.

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General E. coli Culture Conditions

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Media, chemicals, and culture conditions General E. coli cultures were grown in Lysogeny Broth (LB) Miller medium (BD Biosciences, USA) at 37 °C. When indicated, E. coli was also grown on EZ-RICH medium (Teknova, Hollister, CA) supplemented with 1% glucose. Cultures were supplemented with kanamycin (50 mg/L, Sigma Aldrich, USA), or carbenicillin (100mg/L, Sigma Aldrich, USA), when indicated. All other compounds were purchased through Sigma Aldrich (Sigma Aldrich, USA) .

Thompson M.G., Pearson A.N., Barajas J.F., Cruz-Morales P., Sedaghatian N., Costello Z., Garber M.E., Incha M.R., Valencia L.E., Baidoo E.E.K., Martin H.G., Mukhopadhyay A, & Keasling J.D. (2020). Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida. ACS synthetic biology, 9(1).

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