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Team:BYU Provo/Notebook/Auxotrophy/febapr
From 2014.igem.org
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<h3>March 21, 2014</h3> | <h3>March 21, 2014</h3> | ||
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<h2>Week of March 23rd, 2014</h2> | <h2>Week of March 23rd, 2014</h2> | ||
<h3>March 24, 2014</h3> | <h3>March 24, 2014</h3> | ||
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| - | <p>-- | + | <p>--CB TR-- Continued researching auxotrophy ideas.</p> |
<h3>March 26, 2014</h3> | <h3>March 26, 2014</h3> | ||
| - | <p>-- | + | <p>--CB TR-- Presented the circuits for our group, received helpful feedback</p> |
<h3>March 28, 2014</h3> | <h3>March 28, 2014</h3> | ||
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| - | <p>-- | + | <p>--CB TR-- Reviewed literature about the genes involved in Auxotrophy. Also started looking into promoters to use for these genes.</p> |
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<h2>Week of March 27th</h2> | <h2>Week of March 27th</h2> | ||
Revision as of 18:23, 29 September 2014
BYU 2014 Notebook |
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Week of March 9th
March 12, 2014
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
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
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
--CS-- 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
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)
U.S. Outpatient Antibiotic Prescribing, 2010
March 21, 2014
Week of March 23rd, 2014
March 24, 2014
--CB TR-- Continued researching auxotrophy ideas.
March 26, 2014
--CB TR-- Presented the circuits for our group, received helpful feedback
March 28, 2014
--CB TR-- Reviewed literature about the genes involved in Auxotrophy. Also started looking into promoters to use for these genes.
Week of March 27th
March 31, 2014
--CS-- Reviewed more literature about the denitrification genes. Used NCBI BLASTn to confirm that the denitrification genes from Pseudomonas aeruginosa or homologues are not in the N. multiformis genome; only one of the genes (qnorB) has an E value of any significance (3e-22). Located the different denitrification genes in the Pseudomonas aeruginosa PAO1 genome.
April 1, 2014
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.
April 2, 2014
--CS-- Reviewed more literature about denitrification. Found that P. aeruginosa, N. multiformis, and E. coli are all gram negative, so the nitric oxide reductase, which works in the periplasm, should theoretically work in all three bacteria. Identified nirS, norB, norC, and nosZ as the genes needed to insert the denitrification pathway into N. multiformis. Also found that the enzyme that converts nitrite to nitrate in the nitrification process of N. multiformis does so in a reversible reaction, so inserting these genes should force equilibrium through to nitrogen gas.
Differentiated Response of Denitrifying Communities to Fertilization Regime in Paddy Soil.
April 3, 2014
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.
Differentiated Response of Denitrifying Communities to Fertilization Regime in Paddy Soil.
April 4, 2014
Checked denitrifying genes for internal restriction enzyme sequences.
Week of April 12th, 2014
April 7, 2014
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:
- 5’-CCGACCACGTACTGAAGGCCCATGATC-3’
- 5’-GATCATGGGCCTTCTGTACGTGGTCGG-3’
- 5’-TGCAGCCAGTCCTGTAGCACCCCG-3’
- 5’-CGGGGTGCTACAGGTCTGGCTGCA-3’
--CS-- Designed primer sequences for denitrification genes. Forward primers include 3 hanging nucleotides, the XbaI restriction site, and the first several nucleotides of the gene. Reverse primers include 3 hanging nucleotides, the SpeI restriction site, and the last several nucleotides of the gene. The primers were:
- nirS Forward: 5’-CCGTCTAGATGCCATTTGGCAAGCCACTGGTG-3’
- nirS Reverse: 5’-CCGACTAGTTCAGTACACGTCGTGCTGGGTGTT-3’
- norB Forward: 5’-CCGTCTAGATGATGTCGCCCAATGGCTCCCTGA-3’
- norB Reverse: 5’-CCGACTAGTTCAGGCGGCCGCCTTGCCGCGCCGG-3’
- norC Forward: 5’-CCGTCTAGATGTCCGAGACCTTTACCAAAGGC-3’
- norC Reverse: 5’-CGGACTAGTTCAACCCTCCTTGTTCGGCGGCCA-3’
- nosZ Forward: 5’-CCGTCTAGATGAGCGACGACACGAAAAGCCCCC-3’
- nosZ Reverse: 5’-CCGACTAGTTCAAGCCTTTTCCACCAGCATCCGC-3’
Also began researching assay techniques to use in testing the different steps of denitrification.
April 9, 2014
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 E.coli.
--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 E.coli.
April 11, 2014
Performed plasmid preps with the transformed bacteria according to our Common Procedures.
--CS-- Performed plasmid preps for BBa_J23104, BBa_J23105, and BBa_J23106.
Week of April 19th
April 14, 2014
--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.
Week of April 26th
April 21, 2014
Our team prepared a semester final on our N.multiformis metabolism optimization processes.
