Part:BBa_K1442031

Split Neomycin

The split neomycin system consists of both neo-α and neo-β. Neomycin confers kanamycin and geneticin resistance in E. coli and eukaryotic cells, respectively. The split neomycin system has been previously used in Schmidt et al., 2012. Traditionally, antibiotic selection of bacteria is performed with a single antibiotic resistance gene encoded within a single plasmid. If multiple plasmids are introduced into a bacteria, multiple antibiotics are required to select for successful transformants. In addition, the use of multiple antibiotics for selection also stresses the bacteria, which can reduce transformation efficiency.

The split neomycin system overcomes these limitations, by having the α and β subunits encoded on two separate plasmids. Antibiotic resistance is only conferred following heterodimerization of the α and β subunit. Heterodimerization is mediated by leucine zippers, which, as the name implies acts as a zip that joins both the α and β subunits together. This part is useful for the synthetic biology community, as it can save on costs in respect to the purchase of multiple antibiotics.

Vilnius-Lithuania iGEM 2017 Contribution

Our team split the sequence of neomycin resistance protein introduced by iGEM14_Warwick into Alpha-neomycin resistance protein Part:BBa K2259018 and Beta-neomycin resistance protein Part:BBa_K2259019, this modification enables to use this resistance gene in two different plasmids as opposed to a single gene of Part:BBa K1442031, therefor allowing to use it as intended. Besides that, termination codon at the end of the alpha subunit were added to terminate the translation after the peptide is synthesized and leucine zipper domains were removed, making the system lighter, as the heterodimerization occurs naturally.