We have decided to use five different promoters to drive expression of our pili proteins. The T7 promoter (BBa_K1614000) was chosen as it is a strong, well characterised promoter but as this could only be used in BL21(DE3) it was only used for initial expression studies. Three inducible promoters were chosen to allow control over expression in non pili-producing strains of E. coli: the rhamnose inducible promoter P_Rha (BBa_K902065); the arabinose inducible promoter P_ara (BBa_I13453) and the IPTG inducible T5 promoter (pQE30-Qiagen). Finally the constitutive promoter P_J23100 (BBa_J23100) was chosen.
We chose the well characterised strong RBS BBa_B0034 and the double terminator BBa_B0015.
All parts were synthesised as gBlocks by IDT and composite parts were built using a one-pot cloning method protocol modified from Weber et al 2011. All FimH constructs were inserted into pSB1A3 and the fim operon constructs were inserted into pX1’6’0’0 (lab built plasmid). Despite multiple efforts not all of the planned constructs could be built, but the ones that were successful, listed below, were submitted to iGEM in pSB1C3.
FimH Fusion Proteins
Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marrillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One 6, e16765