Team:Exeter/Composite Part

Composite Parts

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 sfGFP

Name Description Base Pairs
BBa_K2324007 P_Rha_FimH_1_sfGFP 2020
BBa_K2324006 P_Rha_FimH_225_sfGFP 2020
BBa_K2324008 P_Rha_FimH_258_sfGFP 2020
BBa_K2324011 P_T7_FimH_225sfGFP 1798

FimH Fusion Proteins

Name Description Base Pairs
BBa_K2324013 P_Rha_FimH_1_His 1102
BBa_K2324009 P_Rha_FimH_1_SynMT 1474
BBa_K2324010 P_Rha_FimH_1_MouseMT 1489

Fim Operon

Name Description Base Pairs
BBa_K2324012 P_J23100_Fim_Operon 5944
BBa_K2324015 P_Ara_Fim_Operon 5874


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