Team:BYU Provo/Notebook/Auxotrophy/febapr

From 2014.igem.org

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<h2 style="003468">Week of March 9th</h2>
<h2 style="003468">Week of March 9th</h2>
<h3>March 12, 2014</h3>
<h3>March 12, 2014</h3>
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<p>Researched possible kill switch options for <i>N. multiformis</i>. Focused on the environment of the bio-reactor to control spread of <i>N. multiformis</i>.</p>
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<p>--CB TR-- Researched possible kill switch options for <i>N. multiformis</i>. Focused on the environment of the bio-reactor to control spread of <i>N. multiformis</i>.</p>
<p>Originally we considered designing a trigger that is specific to the environment of the bioreactor that, when removed from the environment would express a gene that would kill the bacteria. However we realized that such a design would be problematic due to the high mutation rate of <i>N. multiformis</i>. </p>
<p>Originally we considered designing a trigger that is specific to the environment of the bioreactor that, when removed from the environment would express a gene that would kill the bacteria. However we realized that such a design would be problematic due to the high mutation rate of <i>N. multiformis</i>. </p>
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<h2>Week of March 16th</h2>  
<h2>Week of March 16th</h2>  
<h3>March 17, 2014</h3>
<h3>March 17, 2014</h3>
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<p>Researched articles on the environment of waste water treatment plants and the possible genes we could knock out to prepare for our presentation on <i>N.multiformis</i> metabolism optimization.
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<p>--CB TR-- Researched articles on the environment of waste water treatment plants and the possible genes we could knock out to prepare for our presentation on <i>N.multiformis</i> metabolism optimization.
<p>Focused our efforts on a unique gene knock out that would make <i>N. multiformis</i> reliant on the environment of the bio-reactor to grow.</p>
<p>Focused our efforts on a unique gene knock out that would make <i>N. multiformis</i> reliant on the environment of the bio-reactor to grow.</p>
<h3>March 18, 2014</h3>
<h3>March 18, 2014</h3>
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<p>Reviewed all of our literature findings so far. In doing so, identified the specific focuses for our group: inserting the denitrification genes into <i>N. multiformis</i>, making <i>N. multiformis</i> more resistant to pH changes, and making <i>N. multiformis</i> more resistant to heavy metals.</p>
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<p>--CB TR-- Reviewed all of our literature findings so far. In doing so, identified the specific focuses for our group: inserting the denitrification genes into <i>N. multiformis</i>, making <i>N. multiformis</i> more resistant to pH changes, and making <i>N. multiformis</i> more resistant to heavy metals.</p>
<h3>March 19, 2014</h3>
<h3>March 19, 2014</h3>
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<p>--CS-- Presented our ideas for improving the metabolism of <i>N. multiformis</i> and received feedback from the class on them. Confirmed plan to insert the denitrification pathway into <i>N. multiformis</i>. Decided to forego other original goals and instead insert genes that would break down antibiotics.</p>
+
<p>--CB TR-- Presented our ideas for improving the metabolism of <i>N. multiformis</i> and received feedback from the class on them. Confirmed plan to insert the denitrification pathway into <i>N. multiformis</i>. Decided to forego other original goals and instead insert genes that would break down antibiotics.</p>
<h3>March 20, 2014</h3>
<h3>March 20, 2014</h3>
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<p>Searched for the most commonly prescribed antibiotics in the United States. Top prescribed antibiotics include penicillins and macrolides according to the New England Journal of Medicine (2013) </p>
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<p></p>
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<p><a href="http://www.nejm.org/doi/full/10.1056/NEJMc1212055#t=article"><i>U.S. Outpatient Antibiotic Prescribing, 2010</i></a></p>
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<h3>March 21, 2014</h3>
<h3>March 21, 2014</h3>
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<h3>March 24, 2014</h3>
<h3>March 24, 2014</h3>
<p></p>
<p></p>
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<p>--CB TR-- Continued researching auxotrophy ideas.</p>
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<p>--CB TR-- Continued researching auxtrophy ideas.</p>
<h3>March 26, 2014</h3>
<h3>March 26, 2014</h3>
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<p>--CB TR-- Presented the circuits for our group, received helpful feedback</p>
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<p></p>
<h3>March 28, 2014</h3>
<h3>March 28, 2014</h3>
<p></p>
<p></p>
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<p>--CB TR-- Reviewed literature about the genes involved in Auxotrophy. Also started looking into promoters to use for these genes.</p>
 
<h2>Week of March 27th</h2>
<h2>Week of March 27th</h2>
<h3>March 31, 2014</h3>
<h3>March 31, 2014</h3>
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<p>--CS-- Reviewed more literature about the denitrification genes. Used NCBI BLASTn to confirm that the denitrification genes from <i>Pseudomonas aeruginosa</i> or homologues are not in the <i>N. multiformis genome</i>; only one of the genes (qnorB) has an E value of any significance (3e-22). Located the different denitrification genes in the <i>Pseudomonas aeruginosa</i> PAO1 genome.
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<p></p>
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<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/24165750"><i>Pseudomonas aeruginosa and Achromobacter sp.: nitrifying aerobic denitrifiers have a plasmid encoding for denitrifying functional genes.</i></a></p>
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<h3>April 1, 2014</h3>
<h3>April 1, 2014</h3>
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<p> Contacted the 2013 Technical University of Munich IGEM team to inquire about the EreB plasmid since because the registry said that it was not available. Received a response that the part would be available for 2014. Also contacted IGEM to request the part in the 2014 plate.</p>
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<p></p>
<h3>April 2, 2014</h3>
<h3>April 2, 2014</h3>
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<p>--CS-- Reviewed more literature about denitrification. Found that <i>P. aeruginosa</i>, <i>N. multiformis</i>, and <i>E. coli</i> are all gram negative, so the nitric oxide reductase, which works in the periplasm, should theoretically work in all three bacteria. Identified <i>nirS</i>, <i>norB</i>, <i>norC</i>, and <i>nosZ</i> as the genes needed to insert the denitrification pathway into <i>N. multiformis</i>. Also found that the enzyme that converts nitrite to nitrate in the nitrification process of <i>N. multiformis</i> does so in a reversible reaction, so inserting these genes should force equilibrium through to nitrogen gas.</p>
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<p></p>
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<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/21811796"><i>Differentiated Response of Denitrifying Communities to Fertilization Regime in Paddy Soil.</i></a></p>
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<h3>April 3, 2014</h3>
<h3>April 3, 2014</h3>
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<p>Researched scholarly articles about denitrifying genes to determine which particular enzymes are the most important. The paper describes several experiments with these enzymes in soil denitrifiers, the genes required to denitrify, and the importance of each gene present in soil bacteria.
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<p></p>
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<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/21811796"><i>Differentiated Response of Denitrifying Communities to Fertilization Regime in Paddy Soil.</i></a></p>
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<h3>April 4, 2014</h3>
<h3>April 4, 2014</h3>
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<p>Checked denitrifying genes for internal restriction enzyme sequences.</p>
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<p></p>
<h2>Week of April 12th, 2014</h2>
<h2>Week of April 12th, 2014</h2>
<h3> April 7, 2014</h3>
<h3> April 7, 2014</h3>
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<p>Prepared primer sequences to perform mutagenesis to exchange nucleotides and change the restriction site within the gene. Primers were designed for the denitrification norB gene that contained the IGEM plasmid restriction site EcoR1. Those primers were:<ul>
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<p></p>
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<li>5’-CCGACCACGTACTGAAGGCCCATGATC-3’</li>
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<li>5’-GATCATGGGCCTTCTGTACGTGGTCGG-3’</li>
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<li>5’-TGCAGCCAGTCCTGTAGCACCCCG-3’</li>
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<li>5’-CGGGGTGCTACAGGTCTGGCTGCA-3’</li></ul></p>
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<p>--CS-- Designed primer sequences for denitrification genes. Forward primers include 3 hanging nucleotides, the <i>XbaI</i> restriction site, and the first several nucleotides of the gene. Reverse primers include 3 hanging nucleotides, the <i>SpeI</i> restriction site, and the last several nucleotides of the gene. The primers were:<ul>
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<li><i>nirS</i> Forward: 5’-CCGTCTAGATGCCATTTGGCAAGCCACTGGTG-3’</li>
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<li><i>nirS</i> Reverse: 5’-CCGACTAGTTCAGTACACGTCGTGCTGGGTGTT-3’</li>
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<li><i>norB</i> Forward: 5’-CCGTCTAGATGATGTCGCCCAATGGCTCCCTGA-3’</li>
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<li><i>norB</i> Reverse: 5’-CCGACTAGTTCAGGCGGCCGCCTTGCCGCGCCGG-3’</li>
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<li><i>norC</i> Forward: 5’-CCGTCTAGATGTCCGAGACCTTTACCAAAGGC-3’</li>
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<li><i>norC</i> Reverse: 5’-CGGACTAGTTCAACCCTCCTTGTTCGGCGGCCA-3’</li>
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<li><i>nosZ</i> Forward: 5’-CCGTCTAGATGAGCGACGACACGAAAAGCCCCC-3’</li>
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<li><i>nosZ</i> Reverse: 5’-CCGACTAGTTCAAGCCTTTTCCACCAGCATCCGC-3’</li></ul></p>
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<p>Also began researching assay techniques to use in testing the different steps of denitrification.</p>
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<h3>April 9, 2014</h3>
<h3>April 9, 2014</h3>
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<p>Finished the circuit write up for macrolide degradation and outlined a protocol to test the function of the gene. Following the write-up, we transformed the IGEM constitutive promoter BBa_J23109 to test its functionality in competent <i>E.coli</i>.</p>
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<p></p>
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<p>--CS-- Searched online for more assay techniques to use in testing the denitrification genes. Transformed the IGEM constitutive promoters BBa_J23117 and BBa_J23118 into chemically competent <i>E.coli</i>.</p>
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<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/21833336"><i>Regulation and Function of Versatile Aerobic and Anaerobic Respiratory Metabolism in Pseudomonas aeruginosa.</i></a></p>
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<h3> April 11, 2014</h3>
<h3> April 11, 2014</h3>
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<p>Performed plasmid preps with the transformed bacteria according to our <a href="https://2014.igem.org/Team:BYU_Provo/Notebook/CommonProcedures"> Common Procedures</a>.</p>
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<p></p>
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<p>--CS-- Performed plasmid preps for BBa_J23104, BBa_J23105, and BBa_J23106.</p>
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<h2>Week of April 19th</h2>
<h2>Week of April 19th</h2>
<h3> April 14, 2014</h3>
<h3> April 14, 2014</h3>
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<p>--CS-- Decided that instead of finding various assays to test the denitrification genes individually after they have been cloned and transformed, we would first combine all of the denitrification genes into a single plasmid, transform it into the bacteria, and then test the ability of the bacteria to convert nitrate to nitrogen.
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<p> </p>
<h2>Week of April 26th</h2>
<h2>Week of April 26th</h2>
<h3>April 21, 2014</h3>
<h3>April 21, 2014</h3>
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<p> Our team prepared a semester final on our <i>N.multiformis</i> metabolism optimization processes.</p>
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<p> Our team prepared a semester final on our <i>N.multiformis</i> Serine Auxotrophy Process.</p>
</html>
</html>

Revision as of 18:30, 29 September 2014


BYU 2014 Notebook

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Week of March 9th

March 12, 2014

--CB TR-- Researched possible kill switch options for N. multiformis. Focused on the environment of the bio-reactor to control spread of N. multiformis.

Originally we considered designing a trigger that is specific to the environment of the bioreactor that, when removed from the environment would express a gene that would kill the bacteria. However we realized that such a design would be problematic due to the high mutation rate of N. multiformis.

Week of March 16th

March 17, 2014

--CB TR-- Researched articles on the environment of waste water treatment plants and the possible genes we could knock out to prepare for our presentation on N.multiformis metabolism optimization.

Focused our efforts on a unique gene knock out that would make N. multiformis reliant on the environment of the bio-reactor to grow.

March 18, 2014

--CB TR-- Reviewed all of our literature findings so far. In doing so, identified the specific focuses for our group: inserting the denitrification genes into N. multiformis, making N. multiformis more resistant to pH changes, and making N. multiformis more resistant to heavy metals.

March 19, 2014

--CB TR-- Presented our ideas for improving the metabolism of N. multiformis and received feedback from the class on them. Confirmed plan to insert the denitrification pathway into N. multiformis. Decided to forego other original goals and instead insert genes that would break down antibiotics.

March 20, 2014

March 21, 2014

Week of March 23rd, 2014

March 24, 2014

--CB TR-- Continued researching auxtrophy ideas.

March 26, 2014

March 28, 2014

Week of March 27th

March 31, 2014

April 1, 2014

April 2, 2014

April 3, 2014

April 4, 2014

Week of April 12th, 2014

April 7, 2014

April 9, 2014

April 11, 2014

Week of April 19th

April 14, 2014

Week of April 26th

April 21, 2014

Our team prepared a semester final on our N.multiformis Serine Auxotrophy Process.

Retrieved from "http://2014.igem.org/Team:BYU_Provo/Notebook/Auxotrophy/febapr"