Part:BBa_M50082:Design
Encodes PchB and PchA which allows for salicylic acid production in E. coli
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 524
Illegal NgoMIV site found at 928
Illegal NgoMIV site found at 1053
Illegal NgoMIV site found at 1064
Illegal NgoMIV site found at 1345
Illegal NgoMIV site found at 1813 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1995
Design Notes
We did not change the DNA of any of the individual, basic parts in this composite part.
T5 Promoter (M50075): We begin with a strong, constitutive, IPTG inducible T5 promoter obtained from Kara Roger's entry in the Stanford BIOE44 iGEM parts page. We selected a strong promoter because we want to express as much of our final compound as possible. Additionally we chose the IPTG inducible promoter as we can grow our E. Coli in varying ranges of IPTG induction which is essential to experimental design. With genes induced at varying levels, we can detect different levels of output of salicylic acid. RBS Binding Site (M50080): We followed the promoter with a standard strong RBS obtained from Kara Roger's entry in the Stanford BIOE44 iGEM parts page. We selected a strong RBS because we wanted to ensure that the RBS was as functional as possible in order to produce the maximum amount of salicylic acid from the experiment. His Tag (BBa_K112703): Next we encoded for the PchB protein, preceding it with a His tag so that we could detect presence of the protein in a western blot. The His tag was also obtained from parts.igem.org, the sequence was entered by Bing Xia of UC Berkeley, and is unmodified except for its fusion with PchB. Isochorismate Pyruvate-lyase Actuator PchB (BBa_J45017): PchB is an enzyme originally found in Pseudomonas aeruginosa. It naturally catalyzes the conversion of isochorismate to salicylate and pyruvate. The gene for the PchB protein was obtained from the Registry of Biological Parts associated with iGEM. PchB is listed under Part:BBa_J45017 within the iGEM database. The 2006 MIT iGEM team had already codon optimized the gene originally found in P. aeruginosa for use in E. coli. We confirmed codon optimization using the codon optimization software on “Integrated DNA Technologies”. https://www.idtdna.com/CodonOpt Isochorismate Synthase Actuator PchA (BBa_J45017): Following the gene for PchB we encoded the gene for PchA. PchA is also originally found in P. aeruginosa. The gene for PchA was also obtained from the Registry of Biological Parts associated with iGEM and has been codon optimize for use in E. coli by the 2006 MIT iGEM team, and confirmed by our own research. PchA is jointly listed as Part:BBa_J45017 alongside PchB within the iGEM database. The sequence for the PchA protein overlaps with the end of the sequence for PchB. However despite this overlap the two proteins will develop independently and will not be fused together. Located at the end of the PchB coding region is the RBS that corresponds to our PchA. This RBS was also taken from the Registry of Biological Parts associated with iGEM. The RBS is a standard strong RBS as well and was selected for similar reasons to the RBS used with the PcHB. The PchA actuator encodes for a protein that converts chorismate to isochorismate. We found that chorismate is a metabolic precursor found naturally within E. Coli, our selected bacterium. From that we decided that using the PchA protein we could convert it to isochorismate which could then be converted to our desired product, salicylic acid . Flag Tag (BBa_T2004): To the end of the PchA protein we appended a region encoding for a Flag protein tag. When the proteins are translated, the Flag tag will fuse onto the PchA protein and allow it to be located by antibodies in a western blot, further explained in methods and results. The sequence for the flag tag was obtained on parts.igem.org. It was submitted under number BBa_T2004 by Reshma Shetty of Andrew Endy's lab. Terminator (BBa_M50060): Additionally, we encoded for a Phage T7 terminator sequence obtained from Kara Roger's entry in the Stanford BIOE44 iGEM parts page after the PcHA and Flag tag sequences to end transcription.
Source
http://partsregistry.org/Part:BBa_J45017
https://parts.igem.org/wiki/index.php?title=Part:BBa_M50060
https://parts.igem.org/wiki/index.php?title=Part:BBa_M50075
https://parts.igem.org/wiki/index.php?title=Part:BBa_M50080
https://parts.igem.org/wiki/index.php?title=Part:BBa_K112703
https://parts.igem.org/wiki/index.php?title=Part:BBa_T2004
Pseudomonas aeruginosa
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Asirvatham, Margaret. “O638: Identification of Phenols – Ferric Chloride Test.” Lecture Demonstration Manual General Chemistry, University of Colorado-Boulder, 23 Dec. 2015.www.colorado.edu/lab/lecture-demo-manual/o638-identification-phenols-ferric-chloride-test.
Levine, Joseph, and John D. Weber. “Determination of Free Salicylic Acid in Buffered Aspirin Tablets.” Journal of Pharmaceutical Sciences, vol. 57, no. 4, 1968, pp. 631–633., doi:10.1002/jps.2600570418.
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