Difference between revisions of "Team:Cornell/wetlab"
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− | <h1 | + | <h1> <span id = "bio"></span>BioBricks</h1> |
<p>Our project relied heavily on the use of BioBricks. We are in the process of creating 20 different BioBricks for each isoform of EcnB. We have also added our EcnB peptide downstream of stabilization proteins MBP and EDA to help stabilize EcnB production. </p> | <p>Our project relied heavily on the use of BioBricks. We are in the process of creating 20 different BioBricks for each isoform of EcnB. We have also added our EcnB peptide downstream of stabilization proteins MBP and EDA to help stabilize EcnB production. </p> | ||
<p>Our constructs have been summarized as follows: </p> | <p>Our constructs have been summarized as follows: </p> | ||
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− | <h1 | + | <h1> <span id = "chassis" ></span>Chassis</h1> |
BL21 is a cell strain commonly used with the T7 bacteriophage promoter system. In its chromosomal DNA is the T7 RNA polymerase gene, which can be regulated by arabinose induction and glucose inhibition of the araBAD promoter. This allows for efficient and high-level protein expression. Furthermore, the T7 Lysozyme gene in the pLysS plasmid is able to reduce basal expression by suppressing T7 RNA polymerase activity in uninduced cells [1]. | BL21 is a cell strain commonly used with the T7 bacteriophage promoter system. In its chromosomal DNA is the T7 RNA polymerase gene, which can be regulated by arabinose induction and glucose inhibition of the araBAD promoter. This allows for efficient and high-level protein expression. Furthermore, the T7 Lysozyme gene in the pLysS plasmid is able to reduce basal expression by suppressing T7 RNA polymerase activity in uninduced cells [1]. | ||
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− | <h1 id = "isoforms">EcnB Isoform & Strain List</h1> | + | <h1> <span id = "isoforms"></span>EcnB Isoform & Strain List</h1> |
<p>Each of our 20 BioBrick isoforms of the EcnB polypeptide toxin is naturally produced by a different species of bacterium as shown in the chart below. </p> | <p>Each of our 20 BioBrick isoforms of the EcnB polypeptide toxin is naturally produced by a different species of bacterium as shown in the chart below. </p> | ||
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− | <h1 id = "growth"><i><b>Flavobacterium</b></i> Growth </h1> | + | <h1> <span id = "growth"></span><i><b>Flavobacterium</b></i> Growth </h1> |
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− | <h1 id = "stab">Protein Stabilization</h1> | + | <h1> <span id = "stab"></span>Protein Stabilization</h1> |
<p>Because ecnB peptide has a relatively small size of approximately 5.3 kDa (or ~48 amino acids), it can be easily degraded within E. coli after inducing overexpression. To avoid this, we introduce the usage of fusion proteins for enhanced stability and yield. </p> | <p>Because ecnB peptide has a relatively small size of approximately 5.3 kDa (or ~48 amino acids), it can be easily degraded within E. coli after inducing overexpression. To avoid this, we introduce the usage of fusion proteins for enhanced stability and yield. </p> | ||
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− | <h1 id = "zoi">Results</h1> | + | <h1> <span id = "zoi"></span>Results</h1> |
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− | <h1 id="future">Future Work </h1> | + | <h1> <span id="future"></span>Future Work </h1> |
<p>The Cornell iGEM wet lab subteam is committed to discovering the next generation of medicine for fish. Our goal is to provide our customers the most effective and cost-efficient form of flavocide. To accomplish this, we seek to perform a series of medical trials to further test our product's efficacy and we aim to streamline the manufacturing process for large-scaled production.</p> | <p>The Cornell iGEM wet lab subteam is committed to discovering the next generation of medicine for fish. Our goal is to provide our customers the most effective and cost-efficient form of flavocide. To accomplish this, we seek to perform a series of medical trials to further test our product's efficacy and we aim to streamline the manufacturing process for large-scaled production.</p> | ||
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− | <h1 id="refs">References </h1> | + | <h1 > <span id="refs"></span>References </h1> |
<p>[1] Garcia, L., & Molineux, I. (1995). Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli. Journal of Bacteriology, 177(14), 4066-4076.</p><p> [2] Stelzmueller, I., Biebl, M., Wiesmayr, S., Eller, M., Hoeller, E., Fille, M., Weiss, G., Lass-Floerl, C. and Bonatti, H. (2006), Ralstonia pickettii—innocent bystander or a potential threat?. Clinical Microbiology and Infection, 12: 99–101. </p><p> [3] Kittichotirat, W., Good, N., Hall, R., Bringel, F., Lajus, A., Medigue, C., . . . Kalyuzhnaya, M. (2011). Genome Sequence of Methyloversatilis universalis FAM5T, a Methylotrophic Representative of the Order Rhodocyclales. Journal of Bacteriology, 193(17), 4541-4542. doi:10.1128/JB.05331-11 </p><p> [4] Boudon, S., Manceau, C., & Nottéghem, J. (2005). Structure and Origin of Xanthomonas arboricola pv. pruni Populations Causing Bacterial Spot of Stone Fruit Trees in Western Europe. Phytopathology, 95(9), 1081-1088. </p><p> [5] Gai, Z., Wang, X., Tang, H., Tai, C., Tao, F., Wu, G., & Xu, P. (2011). Genome Sequence of Sphingobium yanoikuyae XLDN2-5, an Efficient Carbazole-Degrading Strain. Journal of Bacteriology, 193(22), 6404-6405. doi:10.1128/JB.06050-11 </p><p> [6] Kersters, K., Hinz, K., Hertle, A., Segers, P., Lievens, A., Siegmann, O., & Ley, J. (1984). Bordetella avium sp. nov., Isolated from the Respiratory Tracts of Turkeys and Other Birds. International Journal of Systematic Bacteriology, 34(1), 56-70. doi:10.1099/00207713-34-1-56 </p><p> [7] Holguin, G., Patten, C., & Glick, B. (1999). Genetics and molecular biology of Azospirillum. Biology and Fertility of Soils, 29(1), 10-23. doi:10.1007/s003740050519 </p><p> [8] Stehr-Green, J. K., Centers for Disease Control and Prevention, & National Institutes of Health. (2000). Foodborne disease outbreak investigation: epidemiologic case studies. In Foodborne disease outbreak investigation: epidemiologic case studies. Department of Health & Human Services. </p><p> [9] Enterobacter aerogenes. (2011, April 22). Retrieved August 1, 2015, from https://microbewiki.kenyon.edu/index.php/Enterobacter_aerogenes </p><p> [10] Rice, J., Carrasco-Medina, L., Hodgins, D., & Shewen, P. (2007). Mannheimia haemolytica– and Pasteurella multocida–Induced Bovine Pneumonia. Food Animal Practice, 8(2), 117-28. doi:10.1017/S1466252307001375 [19] Mannheimia haemolytica. (2012, July 18). Retrieved August, 2015, from https://en.wikivet.net/Mannheimia_haemolytica </p><p> [11] Farmer, J.J., Sheth, N., Hudzinski, J., Rose, Harold. Asbury, M. (1982). Bacteremia due to Leptotrichia trevisanii sp. nov. European Journal of Clinical Microbiology & Infectious Diseases, 16(4), 775-778. Retrieved August. 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC272471/ </p><p> [12] Darby, A., Lertpiriyapong, K., Sarkar, U., Seneviratne, U., Park, D., Gamazon, E., . . . Fox, J. (2014). Cytotoxic and Pathogenic Properties of Klebsiella oxytoca Isolated from Laboratory Animals. PLoS ONE. doi:10.1371/journal.pone.0100542</p><p> [13] Sorokin, D. (2005). Thioclava pacifica gen. nov., sp. nov., a novel facultatively autotrophic, marine, sulfur-oxidizing bacterium from a near-shore sulfidic hydrothermal area. International Journal Of Systematic And Evolutionary Microbiology, 1069-1075. Retrieved August 1, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/15879235 </p><p> [14] Escherichia coli. (2014, November 13). Retrieved September 15, 2015, from https://microbewiki.kenyon.edu/index.php/Escherichia_coli</p><p> [15] Howard, A., O’Donoghue, M., Feeney, A., & Sleator, R. (2012, May 1). Acinetobacter baumannii: An emerging opportunistic pathogen. Retrieved August 1, 2015. [ ] Rice, L. (2008). Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. The Journal of Infectious Diseases J INFECT DIS, 197(8), 1079-1081. </p><p> [16] Escherichia coli. (2015). Retrieved August 1, 2015, from https://microbewiki.kenyon.edu/index.php/Escherichia_coli </p><p> [17] Psychrobacter. (2015). Retrieved September 15, 2015, from https://microbewiki.kenyon.edu/index.php/Psychrobacter </p><p> [18] Van Haute, G. (2003, August 1). Agrobacterium tumefaciens. Retrieved August 1, 2015, from http://users.skynet.be/albert.de.koning/agrobacterium.pdf. </p><p> [19] Lai, Q., Liu, Y., Yuan, J., Du, J., Wang, L., Sun, F., & Shao, Z. (2014). Multilocus Sequence Analysis for Assessment of Phylogenetic Diversity and Biogeography in Thalassospira Bacteria from Diverse Marine Environments. Third Institute of Oceanography State Oceanic Administration, 9(9), 1-11. doi:e106353 </p><p> [20] Johnson, K. (2015). Fire blight of apple and pear. Retrieved August 1, 2015, from http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/FireBlight.aspx </p><p> [21] Rossmann, S., Wilson, P., Hicks, J., Carter, B., Cron, S., Simon, C., . . . Kline, M. (1998, June 1). Isolation of Lautropia mirabilis from Oral Cavities of Human Immunodeficiency Virus-Infected Children. Retrieved September 15, 2015.</p> | <p>[1] Garcia, L., & Molineux, I. (1995). Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli. Journal of Bacteriology, 177(14), 4066-4076.</p><p> [2] Stelzmueller, I., Biebl, M., Wiesmayr, S., Eller, M., Hoeller, E., Fille, M., Weiss, G., Lass-Floerl, C. and Bonatti, H. (2006), Ralstonia pickettii—innocent bystander or a potential threat?. Clinical Microbiology and Infection, 12: 99–101. </p><p> [3] Kittichotirat, W., Good, N., Hall, R., Bringel, F., Lajus, A., Medigue, C., . . . Kalyuzhnaya, M. (2011). Genome Sequence of Methyloversatilis universalis FAM5T, a Methylotrophic Representative of the Order Rhodocyclales. Journal of Bacteriology, 193(17), 4541-4542. doi:10.1128/JB.05331-11 </p><p> [4] Boudon, S., Manceau, C., & Nottéghem, J. (2005). Structure and Origin of Xanthomonas arboricola pv. pruni Populations Causing Bacterial Spot of Stone Fruit Trees in Western Europe. Phytopathology, 95(9), 1081-1088. </p><p> [5] Gai, Z., Wang, X., Tang, H., Tai, C., Tao, F., Wu, G., & Xu, P. (2011). Genome Sequence of Sphingobium yanoikuyae XLDN2-5, an Efficient Carbazole-Degrading Strain. Journal of Bacteriology, 193(22), 6404-6405. doi:10.1128/JB.06050-11 </p><p> [6] Kersters, K., Hinz, K., Hertle, A., Segers, P., Lievens, A., Siegmann, O., & Ley, J. (1984). Bordetella avium sp. nov., Isolated from the Respiratory Tracts of Turkeys and Other Birds. International Journal of Systematic Bacteriology, 34(1), 56-70. doi:10.1099/00207713-34-1-56 </p><p> [7] Holguin, G., Patten, C., & Glick, B. (1999). Genetics and molecular biology of Azospirillum. Biology and Fertility of Soils, 29(1), 10-23. doi:10.1007/s003740050519 </p><p> [8] Stehr-Green, J. K., Centers for Disease Control and Prevention, & National Institutes of Health. (2000). Foodborne disease outbreak investigation: epidemiologic case studies. In Foodborne disease outbreak investigation: epidemiologic case studies. Department of Health & Human Services. </p><p> [9] Enterobacter aerogenes. (2011, April 22). Retrieved August 1, 2015, from https://microbewiki.kenyon.edu/index.php/Enterobacter_aerogenes </p><p> [10] Rice, J., Carrasco-Medina, L., Hodgins, D., & Shewen, P. (2007). Mannheimia haemolytica– and Pasteurella multocida–Induced Bovine Pneumonia. Food Animal Practice, 8(2), 117-28. doi:10.1017/S1466252307001375 [19] Mannheimia haemolytica. (2012, July 18). Retrieved August, 2015, from https://en.wikivet.net/Mannheimia_haemolytica </p><p> [11] Farmer, J.J., Sheth, N., Hudzinski, J., Rose, Harold. Asbury, M. (1982). Bacteremia due to Leptotrichia trevisanii sp. nov. European Journal of Clinical Microbiology & Infectious Diseases, 16(4), 775-778. Retrieved August. 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC272471/ </p><p> [12] Darby, A., Lertpiriyapong, K., Sarkar, U., Seneviratne, U., Park, D., Gamazon, E., . . . Fox, J. (2014). Cytotoxic and Pathogenic Properties of Klebsiella oxytoca Isolated from Laboratory Animals. PLoS ONE. doi:10.1371/journal.pone.0100542</p><p> [13] Sorokin, D. (2005). Thioclava pacifica gen. nov., sp. nov., a novel facultatively autotrophic, marine, sulfur-oxidizing bacterium from a near-shore sulfidic hydrothermal area. International Journal Of Systematic And Evolutionary Microbiology, 1069-1075. Retrieved August 1, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/15879235 </p><p> [14] Escherichia coli. (2014, November 13). Retrieved September 15, 2015, from https://microbewiki.kenyon.edu/index.php/Escherichia_coli</p><p> [15] Howard, A., O’Donoghue, M., Feeney, A., & Sleator, R. (2012, May 1). Acinetobacter baumannii: An emerging opportunistic pathogen. Retrieved August 1, 2015. [ ] Rice, L. (2008). Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. The Journal of Infectious Diseases J INFECT DIS, 197(8), 1079-1081. </p><p> [16] Escherichia coli. (2015). Retrieved August 1, 2015, from https://microbewiki.kenyon.edu/index.php/Escherichia_coli </p><p> [17] Psychrobacter. (2015). Retrieved September 15, 2015, from https://microbewiki.kenyon.edu/index.php/Psychrobacter </p><p> [18] Van Haute, G. (2003, August 1). Agrobacterium tumefaciens. Retrieved August 1, 2015, from http://users.skynet.be/albert.de.koning/agrobacterium.pdf. </p><p> [19] Lai, Q., Liu, Y., Yuan, J., Du, J., Wang, L., Sun, F., & Shao, Z. (2014). Multilocus Sequence Analysis for Assessment of Phylogenetic Diversity and Biogeography in Thalassospira Bacteria from Diverse Marine Environments. Third Institute of Oceanography State Oceanic Administration, 9(9), 1-11. doi:e106353 </p><p> [20] Johnson, K. (2015). Fire blight of apple and pear. Retrieved August 1, 2015, from http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/FireBlight.aspx </p><p> [21] Rossmann, S., Wilson, P., Hicks, J., Carter, B., Cron, S., Simon, C., . . . Kline, M. (1998, June 1). Isolation of Lautropia mirabilis from Oral Cavities of Human Immunodeficiency Virus-Infected Children. Retrieved September 15, 2015.</p> | ||
Revision as of 21:23, 18 September 2015