Design and Construction of a Collection of Modular Plasmids to Facilitate Metabolic Engineering Applications in Escherichia coli

The goal of this project is to simplify and accelerate the process of plasmid design and construction for use in metabolic engineering in the bacterium Escherichia coli. A suite of modular plasmids was designed using modules composed of origins of replication, antibiotic resistances, promoters, ribosomal binding sites, and reporter genes, each flanked with common restriction sites. Three 50 base-pair non-coding spacer units were derived from pBluescript II SK and incorporated into the plasmids between modules.  Each spacer was incorporated into the same location in of each of the five plasmids of the collection. The incorporation of common sequences into all the plasmids of the collection allows for swapping of parts by either a restriction enzyme digestion and subsequent ligation or by a restriction enzyme digest followed by a Gibson assembly. Plasmids were assembled using Gibson assembly of PCR-amplified reporter genes, antibiotic resistances, and origins of replication, and three gBlocks that included spacer segments, promoters, ribosomal binding sites, and transcription terminators. Currently, the assembly and characterization of two plasmids, termed pEcSWAP101 and pEcSWAP201 have been completed. pEcSWAP101 functionally expresses the lacZ gene and kanamycin resistance, as shown by the growth of colonies and the blue product produced on agar plates containing both kanamycin and X-gal. pECSWAP201 expresses a resistance to chloramphenicol and contains the gfp gene, as confirmed by PCR. Agarose gels run with samples of restriction enzyme digestions for all restriction sites incorporated indicated successful plasmid assembly. Sequencing done on the regions between the origin of replication, antibiotic resistance gene, and  reporter gene verifies that these components were assembled as designed. Future work includes the assembly of three more plasmids with different modular parts but the same spacer regions. Additionally, the design can be applied to plasmids for other bacterial or yeast strains. This simpler process for design and construction of bacterial plasmids will accelerate plasmid construction with desired components and facilitate study of regulatory elements on gene expression in vivo.