Team:BYU Provo/Notebook/Metabolism/febapr

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<h1 style="color:#FFFFFF" >BYU 2014 Notebook </h1>
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<h1 style="color:#FFFFFF">BYU 2014 Notebook </h1>
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<br></br>
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<p style="color:#FFFFFF"> <a href="https://2014.igem.org/wiki/index.php?title=Team:BYU_Provo/Notebook/Metabolism/febapr&action=edit"style="color:#FFFFFF">Edit Feb April</a> </p>
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<p style="color:#FFFFFF"> <a href="https://2014.igem.org/wiki/index.php?title=Team:BYU_Provo/Notebook&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
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<h2>Week of May 3rd</h2>
 
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<h3>April 29, 2014</h3>
 
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<p></p>
 
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<h3>May 1, 2014</h3>
 
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<p></p>
 
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<h3>May 2, 2014</h3>
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</table>
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<h2>Week of May 10th</h2>
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<h3>May 5, 2014</h3>
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<p>This week in the lab we started our experiment to remove the sera gene from N multiformis.
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We attempted to gain access to the genomic DNA of N multiformis by boiling the organism for 5 min to lyse the cell, this technique has worked on similar organisms in the past and due to its simplicity we opted for this approach.
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After boiling we added our primers and proceeded to perform PCR.  Then run our PCR product on a gel.
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We did this by adding our DNA samples (both forward primers and reverse primers) and a DNA ladder of known size to an agarose gel that had been stained with ethidium bromide and electrophoresing for 30-40 min.
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After isolating our band of DNA we purified it using a freeze and squeeze method.  This method involves excising a band of DNA from the agarose gel and the gel slice cut into small pieces and placed into a micro centrifuge tube. This tube is then placed in a -20C freezer for 20 minutes then removed and immediately centrifuged at 12,000 for 5 minutes at room temp. Agarose debris is will be forced to the bottom of the cup and our now purified DNA is floating on top ready for removal.
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</p>
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<h3>May 6, 2014</h3>
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<p>We finished out the freeze and squeeze experiment.  We took the agarose gel from the freezer that held our PCR product and centrifuged it for 5 min at max speed the added to the following protocol for a freeze and squeeze to get our purified product.
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Freeze and Sqeeze
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2ul fragment one from PCR
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2ul fragment two from PCR
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4ul 5x buffer
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4ul 5x enhancer
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.5ul dNTP
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.5ul primer on left side of left homology block
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.5ul primer on right side of right homology block
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.2ul Q5 enzyme
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QA 20uL ddH2O
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Because Desi said that doing a freeze and squeeze was probably an unnecessary step we also ran a normal where we do not do a freeze and squeeze, rather we run straight to the SOEing part of it.
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SOEing protocol
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1ul fragment one from PCR
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1ul fragment two from PCR
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4ul 5x buffer
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4ul 5x enhancer
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.5ul dNTP
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.5ul primer on left side of left homology block
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.5ul primer on right side of right homology block
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.2ul Q5 enzyme
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QA 20uL ddH2O
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We got our SOEing product and ran it on a gel (picture taken and to be included once we scan it).
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We also performed a restriction digest and plasmid prep.
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</p>
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<h3>May 8, 2014</h3>
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<p></p>
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<h3>May 9, 2014</h3>
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<p></p>
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<h3>May 10, 2014</h3>
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<p></p>
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<h2>Week of May 17th</h2>
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<h3>May 13, 2014</h3>
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<p></p>
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<h3>May 14, 2014</h3>
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<p></p>
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<h3>May 15, 2014</h3>
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<p></p>
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<h2>Week of May 24th</h2>
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<h3>May 20, 2014</h3>
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<p>The past week I have spent approximately 6+ hours researching funding opportunities for our team and estimating costs.
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Opportunities include
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• Rathnau Instituut Grant
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• Departmental Funding
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• Crowd founding initiatives
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o I have started to build a profile on Experiement.com
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• Various Donors
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Verified our clones via colony PCR
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We did this by taking samples from 8 colonies to test for our clone.  Alongside the 8 colonies we also ran a negative control (just the plasmid with no insert) and a positive control (our original soeing product)
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Protocol:
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16.5 DDH2O
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2.5 REdtaq Buffer
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1 DNTP
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1 Primer A
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1 Primer B
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1.25 Redtaq
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2 Boiled template (colony)
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These we PCR’d and wil check on Thursday.
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13May2014 – 19May2014
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The planned schedule
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Week 1 Ligation, Transform
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Week 2 Conjugate (long time)
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• Grow the transformed ecoli (s17)
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• Grow N multiformis
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• Mix together, turn off the lights, but on some Barry White and wait
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Week Work on getting funding
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* experiment.com
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• Fancy black card man•
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Week4 Select for knock out with Kanamycin
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Select  with sucrose
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Grow in serine rich, low, and no environments
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Thursday:
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Today we took tehe product from out ligtion and transformed it. (protocol to be added)
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We made 3 tubes
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Tube 1: 200uL s17, 1uL of 93.1 ng/uL mini-prep pSR47s
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Tube 2: 200uL of our ligation.
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Tube 3: 300ul of LB, 90ul ddH2O, and 1ul of S17
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• S17  is the bacteria that makes our suicide plasma grow.
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All 3 tubes were placed on a shaker at 37 degrees C.
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Tuesday:
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Today (Tuesday) we did ran a gel of the plasmid prep (low melt) of the plasmid digest and the PCR digest (pics to be uploaded)
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We also did a mini-prep with the pSR47s plasmid.
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And a ligation
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</p>
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<h3>May 21, 2014</h3>
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<p></p>
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<h3>May 22, 2014</h3>
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<p></p>
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<h3>May 23, 2014</h3>
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<p></p>
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<h2>Week of May 31st</h2>
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<h3>May 27, 2014</h3>
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<p></p>
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<h3>May 28, 2014</h3>
 
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<p></p>
 
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<h3>May 29, 2014</h3>
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<h2>Week of March 22nd</h2>
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<p></p>
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<h3>March 17, 2014</h3>
 +
<p>Researched articles on the effects of heavy metals on waste water treatment plants and the effects of heavy metals on bacteria to prepare for our presentation on <i>N.multiformis</i> metabolism optimization. Searched for common bacteria with heavy metal resistance genes that could be possibly insert into our bacterial chassis; finding sequences and reading about success rates in data of those that had been transferred.</p>
 +
<p>--CS-- Researched metabolism options more.</p>
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<h3>May 30, 2014</h3>
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<h3>March 18, 2014</h3>
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<p></p>
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<p>--CS-- 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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<h2>Week of June 7th</h2>
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<h3>March 19, 2014</h3>
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<h3>June 2, 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>
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<p>.</p>
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<h3>June 3, 2014</h3>
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<h3>March 20, 2014</h3>
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<p></p>
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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><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>June 4, 2014</h3>
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<h3>March 21, 2014</h3>
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<p></p>
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<p>Searched articles on the effectiveness of macrolide and beta-lactam degradation enzymes. Researched bacteria with a known gene sequences to degrade both types of antibiotics.</p>
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<p>--CS-- Continued researching denitrification pathway. Discovered that there is a BioBrick for denitrification already in the iGEM registry but that it appears to be incomplete (BBa_K1067006).</p>
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<h3>June 5, 2014</h3>
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<h2>Week of March 29th, 2014</h2>
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<p></p>
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<h3>March 24, 2014</h3>
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<p>Investigated macrolide antibiotic degradation, settling on the ethryomycin esterase as the enzyme. Found part BBa_K1159000 in the IGEM registry which contains the Erythromycin Esterase Type II (EreB) gene that degrades macrolides.</p>
 +
<p>--CS-- Continued researching denitrification ideas.</p>
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<h3>June 5, 2014</h3>
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<h3>March 26, 2014</h3>
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<p></p>
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<p>--CS--Presented the circuits for our group and decided to clone the genes ourselves from <i>Pseudomonas aeruginosa</i> instead of using BBa_K1067006.</p>
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<h2>Week of June 14th</h2>
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<h3>March 28, 2014</h3>
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<p>Used the Anderson Promoter Collection to determine which promoters have the highest rate of expression. Antibiotic degradation genes would need medium to strong expression to be useful to the bacteria</p>
 +
<p>--CS-- Reviewed literature about the genes involved in denitrification. Also started looking into promoters to use for these genes. Decided to use a constitutive promoter with medium expression for now and possibly come back and use a nitrate/nitrite-dependent promoter once everything is working right.</p>
 +
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/18390676"><i>Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment</i></a></p>
 +
<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/10347060"><i>Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.</i></a></p>
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<p><a href="http://www.ncbi.nlm.nih.gov/pubmed/10508942"><i>Comparison of Nitrosospira strains isolated from terrestrial environments.</i></a></p>
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<h3>June 10, 2014</h3>
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<h2>Week of April 5th</h2>
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<p></p>
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<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><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>June 12, 2014</h3>
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<h3>April 1, 2014</h3>
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<p></p>
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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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<h3>June 13, 2014</h3>
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<h3>April 2, 2014</h3>
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<p></p>
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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><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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<h2>Week of June 21st</h2>
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<h3>April 3, 2014</h3>
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<h3>June 17, 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>
 +
<h3>April 4, 2014</h3>
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<p>Checked denitrifying genes for internal restriction enzyme sequences.</p>
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<h3>June 19, 2014</h3>
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<h2>Week of April 12th, 2014</h2>
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<p></p>
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<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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<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>
 +
<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>
 +
<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>
 +
<li><i>norC</i> Forward: 5’-CCGTCTAGATGTCCGAGACCTTTACCAAAGGC-3’</li>
 +
<li><i>norC</i> Reverse: 5’-CGGACTAGTTCAACCCTCCTTGTTCGGCGGCCA-3’</li>
 +
<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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<h2>Week of June 28th</h2>
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<h3>April 9, 2014</h3>
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<h3>June 24, 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>We grew cultures of our final product (this time using the proper primers) and perfored a clone verification. We discovered that we had bands at 1000 base pairs!  Yay!! That means that it works and we are not failures.
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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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Now we need to sequence the DNA and we plan on doing plasmid prep as soon as someone returns the missing reagent to the plasmid prep box.
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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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We presented to the class on Wednesday and also ran a plasmid prep now that we know that reagent 1 is kept in the fridge ☺ 
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Desi was totally awesome and did our sequencing for us so thanks Desi.
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The results are shown below
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RESULTS
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SerA-2F
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AWWGRWCGMWTCTCTWTSYKTCSACGGTATCSATAAGCTTGATATCGAATTCCTGCASCCCGGGGGATCCGGCGTAATGGMACWCATGATCAAAACGGCGGCGGATGCCGAAGCGGCCGTAAACKCAGTATATTACCCTCCACGCGGACAACGTGGAGTCGGGCTTGCACGCGCCCAGGGGTATGGTGCGCGATTTCAGCAATATCGCCACTGGCTGGAAGAAAATGCCGTCATCATTGCCATGATCGAGCATATCGATGCAGTCGATGCCATCGATTCGATTCTTTCCGTTCCGGGAATCGATGCTTATATCATAGGTCCCTACGATCTTTCAGGATCGCTCGGCCGTCCCGGAGAACTCGCTGATCCGGAAGTCCAGGCTGCGGTAGAACGGGTAAAAGATGCCGGGCGACGCGCGGGCAAGGCAGGCGGCATTCATGTAGTCGAACCCAATCCGGAACAATTGCGCCGCAATATCGAGGCGGGCTTCAGTTTTCTTGGCTATGGCCTGGATATCCGCATTCTCGATACCGTCTGCCGCAGTCATCTGCAAAACATCAGGGAAGCTCTATGAACAAACTTGCGATTTCCACCTCGTCATTCGATGTCAGCATCGAAGCCGCTATGGCTCGCTCTATGACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTRTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAWTTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAMCCTGTCGTGCCAGCTGSATTAATGAATCGGCCAACGCTARAWTTCCCATGTCAGCCGTTAAGTGTTCCTGTGTCACTCAAAATTGCTTGARAGGSTCTAARGGCTTCTCARTGCGTTACATCCCTGGSTTGTKGTCCRCACCGTTAAACCTTAAAAGCTTRARAGGCTTATATATCTTTTTTCTWAWAAAMCTAAAACCTARRRGCTATTAGTTGCTGAATTTATATAAGTTAATGGTCARACATGAGAGCTAAGWASGGGAAAMTGGARAGCGTAGTACGTAGCCATGAAAGCTTAGTACGTAGCCAGGAGGGGTTAARGTCGTACATGAAGCTTAGTACGTAACCGTGAGAGCCTATAAMCGTGAAAGCGTGGATARGCCGGAT</p>
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<h3>June 25, 2014</h3>
 
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<p>--CB TR-- Today we prepared our presentations. We then gave them.</p>
 
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<h3>June 26, 2014</h3>
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<h3> April 11, 2014</h3>
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<p>--CB TR-- Today we performed a plasmid prep (see protocol in common procedures section)</p>
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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>
 +
<p>--CS-- Performed plasmid preps for BBa_J23104, BBa_J23105, and BBa_J23106.</p>
 +
<h2>Week of April 19th</h2>
 +
<h3> April 14, 2014</h3>
 +
<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.
 +
<h2>Week of April 26th</h2>
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<h3>April 21, 2014</h3>
 +
<p> Our team prepared a semester final on our <i>N.multiformis</i> metabolism optimization processes.</p>
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</body>
 
</html>
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Revision as of 21:38, 1 October 2014


BYU 2014 Notebook

Edit Feb April

Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

Week of March 22nd

March 17, 2014

Researched articles on the effects of heavy metals on waste water treatment plants and the effects of heavy metals on bacteria to prepare for our presentation on N.multiformis metabolism optimization. Searched for common bacteria with heavy metal resistance genes that could be possibly insert into our bacterial chassis; finding sequences and reading about success rates in data of those that had been transferred.

--CS-- Researched metabolism options more.

March 18, 2014

--CS-- 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

Searched articles on the effectiveness of macrolide and beta-lactam degradation enzymes. Researched bacteria with a known gene sequences to degrade both types of antibiotics.

--CS-- Continued researching denitrification pathway. Discovered that there is a BioBrick for denitrification already in the iGEM registry but that it appears to be incomplete (BBa_K1067006).

Week of March 29th, 2014

March 24, 2014

Investigated macrolide antibiotic degradation, settling on the ethryomycin esterase as the enzyme. Found part BBa_K1159000 in the IGEM registry which contains the Erythromycin Esterase Type II (EreB) gene that degrades macrolides.

--CS-- Continued researching denitrification ideas.

March 26, 2014

--CS--Presented the circuits for our group and decided to clone the genes ourselves from Pseudomonas aeruginosa instead of using BBa_K1067006.

March 28, 2014

Used the Anderson Promoter Collection to determine which promoters have the highest rate of expression. Antibiotic degradation genes would need medium to strong expression to be useful to the bacteria

--CS-- Reviewed literature about the genes involved in denitrification. Also started looking into promoters to use for these genes. Decided to use a constitutive promoter with medium expression for now and possibly come back and use a nitrate/nitrite-dependent promoter once everything is working right.

Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Comparison of Nitrosospira strains isolated from terrestrial environments.

Week of April 5th

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.

Pseudomonas aeruginosa and Achromobacter sp.: nitrifying aerobic denitrifiers have a plasmid encoding for denitrifying functional genes.

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.

Regulation and Function of Versatile Aerobic and Anaerobic Respiratory Metabolism in Pseudomonas aeruginosa.

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.

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