Team:BYU Provo/Notebook/Metabolism/febapr

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BYU 2014 Notebook

Edit February April

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Week of March 15th

March 12, 2014

--CS-- Reviewed literature about N. multiformis. Found that it does have genes for nitrification, the conversion of ammonia to nitrate, but not for denitrificaiton, the conversion of nitrate to nitrogen gas. Putting denitrification genes in it would thus be beneficial.

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) http://www.nejm.org/doi/full/10.1056/NEJMc1212055#t=article

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

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

Week of April 5th

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 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. http://link.springer.com/article/10.1007%2Fs00248-011-9909-5/fulltext.html

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’

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 BBaJ23109 to test its functionality in competent E.coli.

April 11, 2014

Performed plasmid preps with the transformed bacteria according to our Common Procedures.

Week of April 19th

Week of April 26th

April 21, 2014

Our team prepared a semester final on our N.multiformis metabolism optimization processes.