Part:BBa_M50005:Design
alkG
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 352
Design Notes
This plasmid is an actuator designed to produce the alkG protein, the second step in degrading alkanes to clean up oil spills. We designed a plasmid with a high ORF to produce maximum copies of the alkG protein. The plasmid has kanamycin resistance, such that only E. Coli with a copy of this plasmid will grow on kanamycin plates. Note that the alkG plasmid has a different antibiotic than the alkB1 plasmid; this design allows us to select for E. Coli colonies that contain both plasmids on an ampicillin and kanamycin plates. The plasmid has a rhamnose inducible promoter, meaning that rhamnose activates the expression of alkG. Note that the alkB1 plasmid has a different kind of promoter; this design is intended so that we can control the relative amount of expression of each plasmid. We used a strong ribosome binding site for this plasmid because we wanted to have maximal translation of the alkG protein. The alkG gene is encoded after the ribosome binding site, the sequence of which was retrieved using the Kyoto Encyclopedia of Genes and Genomes. The sequence for alkG was revised to codon-optimize for E. Coli in GeneDesigner and some base pairs were modified to eliminate repeats using GeneDesigner. This gene, in tandem with the alkB1 gene, adds a hydroxyl group to an alkane to make it more appropriate for bacterial metabolism (Yakimov et al.). The alkG gene is tagged with the HIS fusion tag, courtesy of DNA 2.0, in order to differentiate its expression from the alkB1 plasmid. The sequences for the ORF, kanamycin resistance, rhamnose promoter, and ribosome binding site all come from DNA 2.0.
Source
alkG comes from the genome of Alcanivorax Borkumensis.
References
“Alcanivorax Borkumensis: ABO_2708.” Kyoto Encyclopedia of Genes and Genomes. N.p., n.d. Web. 31 Oct. 2016.