Team:Penn/VerifiGEM

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<b><div class="name" align="center">Use of the GRAS (Generally Regarded As Safe) Strain E. Coli Nissle 1917</div></b>
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<br>
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<p style="color:black;text-indent:30px;">  sdadfasd</p>
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<p style="color:black;text-indent:30px;">asdfsdfsf</p><div align="center">
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<img src="https://static.igem.org/mediawiki/2012/9/90/20121004033558!IMG_3309.JPG" width="600" height="400"></div>
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<b><div class="name" align="center">Production of Chemically Competent Nissle 1911 Cells</div></b>
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<ol>
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<li>Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C
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overnight.</li>
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<li>Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).</li>
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<li>Shake vigorously at 37 °C to OD600 ~0.25-0.3.</li>
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<li>Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2
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solution and 0.1M CaCl2 plus 15% glycerol are on ice.</li>
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<li>Centrifuge the cells for 10 min at 5000 g at 4°C.</li>
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<li>Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.</li>
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<li>Centrifuge the cells as above.</li>
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<li>Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2
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solution plus 15% glycerol.</li>
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<li>Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.
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<li>
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Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.
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<b><div class="name" align="center">References:</div></b><br>
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[1]    Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.<br>
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<br>
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[2]    Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay.
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"Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.<br>
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<br>
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[3]    Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral
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Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012. <br>
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<br>
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[4]      Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.</div>
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Revision as of 04:08, 26 October 2012

Penn 2012 iGEM Wiki

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Use of the GRAS (Generally Regarded As Safe) Strain E. Coli Nissle 1917

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Production of Chemically Competent Nissle 1911 Cells
  1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight.
  2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
  3. Shake vigorously at 37 °C to OD600 ~0.25-0.3.
  4. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
  5. Centrifuge the cells for 10 min at 5000 g at 4°C.
  6. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.
  7. Centrifuge the cells as above.
  8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
  9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.
    • Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.
References:

[1] Grozdanov, L., U. Zahringer, G. Blum-Oehler, L. Brade, A. Henne, Y. A. Knirel, U.Schombel, J. Schulze, U. Sonnenborn, G. Gottschalk, J. Hacker, E. T. Rietschel, and U. Dobrindt. "A Single Nucleotide Exchange in the Wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia Coli Strain Nissle 1917." Journal of Bacteriology 184.21 (2002): 5912-925. Print.

[2] Stritzker, J., S. Weibel, P. Hill, T. Oelschlaeger, W. Goebel, and A. Szalay. "Tumor-specific Colonization, Tissue Distribution, and Gene Induction by Probiotic Escherichia Coli Nissle 1917 in Live Mice." International Journal of Medical Microbiology 297.3 (2007): 151-62. 19 Apr. 2007. Web. 29 Sept. 2012.

[3] Weise, Christin, Yan Zhu, Dennis Ernst, Anja A. Kühl, and Margitta Worm. "Oral Administration of Escherichia Coli Nissle 1917 Prevents Allergen-induced Dermatitis in Mice." Experimental Dermatology 20.10 (2011): 805-09. 11 July 2011. Web. 29 Sept. 2012.

[4] Zhang, Y., L. Xia, X. Zhang, X. Ding, F. Yan, and F. Wu. "Escherichia Coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumor through the Expression of Azurin Protein." Applied Environmental Microbiology (n.d.): n. pag. 24 Aug. 2012. Web. 29 Sept. 2012.

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