Engineering and Analyzing Multicellular Systems: Methods and by Lianhong Sun, Wenying Shou

By Lianhong Sun, Wenying Shou

Engineering Multicellular platforms: equipment and Protocols, specializes in laboratory systems utilized in fresh efforts for developing artificial multicellular structures and their purposes. specifically, developing multicellular structures to shape a variety of microbial ecosystems has been commonly explored to envision evolution and interactions of microbial ecosystems, whereas co-cultures have emerged as an effective software to provide a few complicated chemical molecules. Written within the hugely profitable Methods in Molecular Biology series layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, quite simply reproducible laboratory protocols and key tips about troubleshooting and heading off identified pitfalls.

Engineering Multicellular structures: tools and Protocols supply a finished laboratory protocol reference for developing multicellular platforms for varied applications.

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Proc Natl Acad Sci U S A 99: 15451–15455 38. Rakoff-Nahoum S, Paglino J, EslamiVarzaneh F et al (2004) Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229–241 39. Hooper LV (2004) Bacterial contributions to mammalian gut development. Trends Microbiol 12:129–134 40. Pryde SE, Duncan SH, Hold GL et al (2002) The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett 217:133–139 41. Round JL, Mazmanian SK (2010) Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota.

8. Autoclaved water, tubes, and tips. 2 Components for Mating, Sporulation, and Tetrad Dissection 1. 2 g/l raffinose, bring to final volume with diH2O. Autoclave. 2. 1 M sodium citrate (see Note 4), 60 mM EDTA. 0 with 38 % HCl (see Note 5). Autoclave using a 20′ sterilization cycle, and remove promptly (see Note 6). 3. Zymolyase 20T: 30 mg zymolyase 20T dissolved in 10 ml SCE (see Note 7). 1 Primer Design and Amplification for Gene Tagging or Replacement 1. Design primers: For gene replacement (Fig.

13. If selection is on complementation of nutrient auxotrophy, cells can be directly plated on selective medium. If selection is on drug resistance, cells need to be incubated for ~2–3 h at 30 °C in 1 ml YPD to express the resistance gene before being plated. 14. To plate, first centrifuge at 3,824 × g for 15 s. Discard 700 μl of the supernatant. Resuspend cells in the remaining 300 μl and plate on 80 % of the surface. Use a sterile toothpick to streak from the plated area to the empty area to maximize the chance of obtaining single colonies.

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